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Sample records for intestinal enzyme acyl

  1. Are There Acyl-Homoserine Lactones within Mammalian Intestines?

    PubMed Central

    Swearingen, Matthew C.; Sabag-Daigle, Anice

    2013-01-01

    Many Proteobacteria are capable of quorum sensing using N-acyl-homoserine lactone (acyl-HSL) signaling molecules that are synthesized by LuxI or LuxM homologs and detected by transcription factors of the LuxR family. Most quorum-sensing species have at least one LuxR and one LuxI homolog. However, members of the Escherichia, Salmonella, Klebsiella, and Enterobacter genera possess only a single LuxR homolog, SdiA, and no acyl-HSL synthase. The most obvious hypothesis is that these organisms are eavesdropping on acyl-HSL production within the complex microbial communities of the mammalian intestinal tract. However, there is currently no evidence of acyl-HSLs being produced within normal intestinal communities. A few intestinal pathogens, including Yersinia enterocolitica, do produce acyl-HSLs, and Salmonella can detect them during infection. Therefore, a more refined hypothesis is that SdiA orthologs are used for eavesdropping on other quorum-sensing pathogens in the host. However, the lack of acyl-HSL signaling among the normal intestinal residents is a surprising finding given the complexity of intestinal communities. In this review, we examine the evidence for and against the possibility of acyl-HSL signaling molecules in the mammalian intestine and discuss the possibility that related signaling molecules might be present and awaiting discovery. PMID:23144246

  2. Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes.

    PubMed

    Soupene, Eric; Fyrst, Henrik; Kuypers, Frans A

    2008-01-01

    The mammalian RBC lacks de novo lipid synthesis but maintains its membrane composition by rapid turnover of acyl moieties at the sn-2 position of phospholipids. Plasma-derived fatty acids are esterified to acyl-CoA by acyl-CoA synthetases and transferred to lysophospholipids by acyl-CoA:lysophospholipid acyltransferases. We report the characterization of three lysophosphatidylcholine (lysoPC) acyltransferases (LPCATs), products of the AYTL1, -2, and -3 genes. These proteins are three members of a LPCAT family, of which all three genes are expressed in an erythroleukemic cell line. Aytl2 mRNA was detected in mouse reticulocytes, and the presence of the product of the human ortholog was confirmed in adult human RBCs. The three murine Aytl proteins generated phosphatidylcholine from long-chain acyl-CoA and lysoPC when expressed in Escherichia coli membranes. Spliced variants of Aytl1, affecting a conserved catalytic motif, were identified. Calcium and magnesium modulated LPCAT activity of both Aytl1 and -2 proteins that exhibit EF-hand motifs at the C terminus. Characterization of the product of the Aytl2 gene as the phosphatidylcholine reacylating enzyme in RBCs represents the identification of a plasma membrane lysophospholipid acyltransferase and establishes the function of a LPCAT protein.

  3. Acylation of salmon calcitonin modulates in vitro intestinal peptide flux through membrane permeability enhancement.

    PubMed

    Trier, Sofie; Linderoth, Lars; Bjerregaard, Simon; Strauss, Holger M; Rahbek, Ulrik L; Andresen, Thomas L

    2015-10-01

    Acylation of peptide drugs with fatty acid chains has proven beneficial for prolonging systemic circulation, as well as increasing enzymatic stability and interactions with lipid cell membranes. Thus, acylation offers several potential benefits for oral delivery of therapeutic peptides, and we hypothesize that tailoring the acylation may be used to optimize intestinal translocation. This work aims to characterize acylated analogues of the therapeutic peptide salmon calcitonin (sCT), which lowers blood calcium, by systematically increasing acyl chain length at two positions, in order to elucidate its influence on intestinal cell translocation and membrane interaction. We find that acylation drastically increases in vitro intestinal peptide flux and confers a transient permeability enhancing effect on the cell layer. The analogues permeabilize model lipid membranes, indicating that the effect is due to a solubilization of the cell membrane, similar to transcellular oral permeation enhancers. The effect is dependent on pH, with larger effect at lower pH, and is impacted by acylation chain length and position. Compared to the unacylated peptide backbone, N-terminal acylation with a short chain provides 6- or 9-fold increase in peptide translocation at pH 7.4 and 5.5, respectively. Prolonging the chain length appears to hamper translocation, possibly due to self-association or aggregation, although the long chain acylated analogues remain superior to the unacylated peptide. For K(18)-acylation a short chain provides a moderate improvement, whereas medium and long chain analogues are highly efficient, with a 12-fold increase in permeability compared to the unacylated peptide backbone, on par with currently employed oral permeation enhancers. For K(18)-acylation the medium chain acylation appears to be optimal, as elongating the chain causes greater binding to the cell membrane but similar permeability, and we speculate that increasing the chain length further may

  4. The Golgi S-acylation machinery comprises zDHHC enzymes with major differences in substrate affinity and S-acylation activity

    PubMed Central

    Lemonidis, Kimon; Gorleku, Oforiwa A.; Sanchez-Perez, Maria C.; Grefen, Christopher; Chamberlain, Luke H.

    2014-01-01

    S-acylation, the attachment of fatty acids onto cysteine residues, regulates protein trafficking and function and is mediated by a family of zDHHC enzymes. The S-acylation of peripheral membrane proteins has been proposed to occur at the Golgi, catalyzed by an S-acylation machinery that displays little substrate specificity. To advance understanding of how S-acylation of peripheral membrane proteins is handled by Golgi zDHHC enzymes, we investigated interactions between a subset of four Golgi zDHHC enzymes and two S-acylated proteins—synaptosomal-associated protein 25 (SNAP25) and cysteine-string protein (CSP). Our results uncover major differences in substrate recognition and S-acylation by these zDHHC enzymes. The ankyrin-repeat domains of zDHHC17 and zDHHC13 mediated strong and selective interactions with SNAP25/CSP, whereas binding of zDHHC3 and zDHHC7 to these proteins was barely detectable. Despite this, zDHHC3/zDHHC7 could S-acylate SNAP25/CSP more efficiently than zDHHC17, whereas zDHHC13 lacked S-acylation activity toward these proteins. Overall the results of this study support a model in which dynamic intracellular localization of peripheral membrane proteins is achieved by highly selective recruitment by a subset of zDHHC enzymes at the Golgi, combined with highly efficient S-acylation by other Golgi zDHHC enzymes. PMID:25253725

  5. Acylated monogalactosyl diacylglycerol: prevalence in the plant kingdom and identification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana.

    PubMed

    Nilsson, Anders K; Johansson, Oskar N; Fahlberg, Per; Kommuri, Murali; Töpel, Mats; Bodin, Lovisa J; Sikora, Per; Modarres, Masoomeh; Ekengren, Sophia; Nguyen, Chi T; Farmer, Edward E; Olsson, Olof; Ellerström, Mats; Andersson, Mats X

    2015-12-01

    The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono- and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl-MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl-MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12-oxo-phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA-containing galactolipids in the plant kingdom. While acyl-MGDG was found to be ubiquitous in green tissue of plants ranging from non-vascular plants to angiosperms, OPDA-containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non-oxidized and OPDA-containing acyl-MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl-MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response. PMID:26566971

  6. Serpin-protease complexes are trapped as stable acyl-enzyme intermediates.

    PubMed

    Lawrence, D A; Ginsburg, D; Day, D E; Berkenpas, M B; Verhamme, I M; Kvassman, J O; Shore, J D

    1995-10-27

    The serine protease inhibitors of the serpin family are an unusual group of proteins thought to have metastable native structures. Functionally, they are unique among polypeptide protease inhibitors, although their precise mechanism of action remains controversial. Conflicting results from previous studies have suggested that the stable serpin-protease complex is trapped in either a tight Michaelis-like structure, a tetrahedral intermediate, or an acyl-enzyme. In this report we show that, upon association with a target protease, the serpin reactive-center loop (RCL) is cleaved resulting in formation of an acyl-enzyme intermediate. This cleavage is coupled to rapid movement of the RCL into the body of the protein bringing the inhibitor closer to its lowest free energy state. From these data we suggest a model for serpin action in which the drive toward the lowest free energy state results in trapping of the protease-inhibitor complex as an acyl-enzyme intermediate. PMID:7592687

  7. Intestinal acyl-CoA synthetase 5: activation of long chain fatty acids and behind.

    PubMed

    Klaus, Christina; Jeon, Min Kyung; Kaemmerer, Elke; Gassler, Nikolaus

    2013-11-14

    The intestinal mucosa is characterized by a high complexity in terms of structure and functions and allows for a controlled demarcation towards the gut lumen. On the one hand it is responsible for pulping and selective absorption of alimentary substances ensuring the immunological tolerance, on the other hand it prevents the penetration of micro-organisms as well as bacterial outgrowth. The continuous regeneration of surface epithelia along the crypt-villus-axis in the small intestine is crucial to assuring these various functions. The core phenomena of intestinal epithelia regeneration comprise cell proliferation, migration, differentiation, and apoptosis. These partly contrarily oriented processes are molecularly balanced through numerous interacting signaling pathways like Wnt/β-catenin, Notch and Hedgehog, and regulated by various modifying factors. One of these modifiers is acyl-CoA synthetase 5 (ACSL5). It plays a key role in de novo lipid synthesis, fatty acid degradation and membrane modifications, and regulates several intestinal processes, primarily through different variants of protein lipidation, e.g., palmitoylation. ACSL5 was shown to interact with proapoptotic molecules, and besides seems to inhibit proliferation along the crypt-villus-axis. Because of its proapoptotic and antiproliferative characteristics it could be of significant relevance for intestinal homeostasis, cellular disorder and tumor development. PMID:24259967

  8. Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes

    PubMed Central

    Chankhamhaengdecha, Surang; Hongvijit, Suphatra; Srichaisupakit, Akkaraphol; Charnchai, Pattra; Panbangred, Watanalai

    2013-01-01

    Several Gram-negative pathogenic bacteria employ N-acyl-L-homoserine lactone (HSL) quorum sensing (QS) system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE) results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9%) and 68 (51.5%) of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at 151.30 ± 3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified as Streptomyces based on 16S  rRNA gene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In an in vitro pathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused by Pectobacterium carotovorum ssp. carotovorum as demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophytic Streptomyces. PMID:23484156

  9. Endophytic actinomycetes: a novel source of potential acyl homoserine lactone degrading enzymes.

    PubMed

    Chankhamhaengdecha, Surang; Hongvijit, Suphatra; Srichaisupakit, Akkaraphol; Charnchai, Pattra; Panbangred, Watanalai

    2013-01-01

    Several Gram-negative pathogenic bacteria employ N-acyl-L-homoserine lactone (HSL) quorum sensing (QS) system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE) results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9%) and 68 (51.5%) of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at 151.30 ± 3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified as Streptomyces based on 16S  rRNA gene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In an in vitro pathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused by Pectobacterium carotovorum ssp. carotovorum as demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophytic Streptomyces.

  10. Tryptophan fluorescence reveals induced folding of Vibrio harveyi acyl carrier protein upon interaction with partner enzymes.

    PubMed

    Gong, Huansheng; Murphy, Peter W; Langille, Gavin M; Minielly, Sarah J; Murphy, Anne; McMaster, Christopher R; Byers, David M

    2008-11-01

    We have introduced tryptophan as a local fluorescent probe to monitor the conformation of Vibrio harveyi acyl carrier protein (ACP), a small flexible protein that is unfolded at neutral pH but must undergo reversible conformational change during the synthesis and delivery of bacterial fatty acids. Consistent with known 3D structures of ACP, steady-state fluorescence and quenching experiments indicated that Trp at positions 46, 50, and 72 are buried in the hydrophobic core upon Mg(2+)-induced ACP folding, whereas residues 25 and 45 remain in a hydrophilic environment on the protein surface. Attachment of fatty acids to the phosphopantetheine prosthetic group progressively stabilized the folded conformation of all Trp-substituted ACPs, but longer chains (14:0) were less effective than medium chains (8:0) in shielding Trp from acrylamide quenching in the L46W protein. Interaction with ACP-dependent enzymes LpxA and holo-ACP synthase also caused folding of L46W; fluorescence quenching indicated proximity of Trp-45 in helix II of ACP in LpxA binding. Our results suggest that divalent cations and fatty acylation produce differing environments in the ACP core and also reveal enzyme partner-induced folding of ACP, a key feature of "natively unfolded" proteins.

  11. Enhanced production of polyunsaturated fatty acids by enzyme engineering of tandem acyl carrier proteins

    PubMed Central

    Hayashi, Shohei; Satoh, Yasuharu; Ujihara, Tetsuro; Takata, Yusuke; Dairi, Tohru

    2016-01-01

    In some microorganisms, polyunsaturated fatty acids (PUFAs) are biosynthesized by PUFA synthases characterized by tandem acyl carrier proteins (ACPs) in subunit A. These ACPs were previously shown to be important for PUFA productivity. In this study, we examined their function in more detail. PUFA productivities increased depending on the number of ACPs without profile changes in each subunit A of eukaryotic and prokaryotic PUFA synthases. We also constructed derivative enzymes from subunit A with 5 × ACPs. Enzymes possessing one inactive ACP at any position produced ~30% PUFAs compared with the parental enzyme but unexpectedly had ~250% productivity compared with subunit A with 4 × ACPs. Enzymes constructed by replacing the 3rd ACP with an inactive ACP from another subunit A or ACP-unrelated sequences produced ~100% and ~3% PUFAs compared with the parental 3rd ACP-inactive enzyme, respectively. These results suggest that both the structure and number of ACP domains are important for PUFA productivity. PMID:27752094

  12. Theoretical approach to the steady-state kinetics of a bi-substrate acyl-transfer enzyme reaction that follows a hydrolysable-acyl-enzyme-based mechanism. Application to the study of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung.

    PubMed Central

    Martín, J; Pérez-Gil, J; Acebal, C; Arche, R

    1990-01-01

    A kinetic model is proposed for catalysis by an enzyme that has several special characteristics: (i) it catalyses an acyl-transfer bi-substrate reaction between two identical molecules of substrate, (ii) the substrate is an amphiphilic molecule that can be present in two physical forms, namely monomers and micelles, and (iii) the reaction progresses through an acyl-enzyme-based mechanism and the covalent intermediate can react also with water to yield a secondary hydrolytic reaction. The theoretical kinetic equations for both reactions were deduced according to steady-state assumptions and the theoretical plots were predicted. The experimental kinetics of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung fitted the proposed equations with great accuracy. Also, kinetics of inhibition by products behaved as expected. It was concluded that the competition between two nucleophiles for the covalent acyl-enzyme intermediate, and not a different enzyme action depending on the physical state of the substrate, is responsible for the differences in kinetic pattern for the two activities of the enzyme. This conclusion, together with the fact that the kinetic equation for the transacylation is quadratic, generates a 'hysteretic' pattern that can provide the basis of self-regulatory properties for enzymes to which this model could be applied. PMID:2310381

  13. Theoretical approach to the steady-state kinetics of a bi-substrate acyl-transfer enzyme reaction that follows a hydrolysable-acyl-enzyme-based mechanism. Application to the study of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung.

    PubMed

    Martín, J; Pérez-Gil, J; Acebal, C; Arche, R

    1990-02-15

    A kinetic model is proposed for catalysis by an enzyme that has several special characteristics: (i) it catalyses an acyl-transfer bi-substrate reaction between two identical molecules of substrate, (ii) the substrate is an amphiphilic molecule that can be present in two physical forms, namely monomers and micelles, and (iii) the reaction progresses through an acyl-enzyme-based mechanism and the covalent intermediate can react also with water to yield a secondary hydrolytic reaction. The theoretical kinetic equations for both reactions were deduced according to steady-state assumptions and the theoretical plots were predicted. The experimental kinetics of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung fitted the proposed equations with great accuracy. Also, kinetics of inhibition by products behaved as expected. It was concluded that the competition between two nucleophiles for the covalent acyl-enzyme intermediate, and not a different enzyme action depending on the physical state of the substrate, is responsible for the differences in kinetic pattern for the two activities of the enzyme. This conclusion, together with the fact that the kinetic equation for the transacylation is quadratic, generates a 'hysteretic' pattern that can provide the basis of self-regulatory properties for enzymes to which this model could be applied. PMID:2310381

  14. Brush border intestinal enzymes after multiple daily fractionation

    SciTech Connect

    Becciolini, A.; Giache, V.; Balzi, M.; Morrone, A.

    1987-03-01

    The modifications in brush border enzyme activity of the epithelial cell of the small intestine were studied after multiple daily fractionation (MDF) of 3 Gy X and 3 Gy X 2 X 2 (12 h split). Disaccharase and dipeptidase activities changed in the same way after irradiation. The results show that both total doses caused the three known phases of increase, decrease, and a return to normal. With MDF, activity at the end of irradiation was similar to or greater than that of controls and remained higher longer than a single dose of 8 Gy. However, the return to normal occurred sooner than after a single dose of 8 Gy. After 11 days, circadian oscillations of brush border enzyme activity appeared similar to those of controls in many segments of the intestine, reaching the highest activity during the night and the lowest in the afternoon.

  15. Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate.

    PubMed

    Maloney, Finn P; Gerwick, Lena; Gerwick, William H; Sherman, David H; Smith, Janet L

    2016-09-13

    Alkyl branching at the β position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a β-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (Kd = 0.5 μM) and could not be substituted by the acceptor ACP. High-resolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre- and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA.

  16. Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate.

    PubMed

    Maloney, Finn P; Gerwick, Lena; Gerwick, William H; Sherman, David H; Smith, Janet L

    2016-09-13

    Alkyl branching at the β position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a β-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (Kd = 0.5 μM) and could not be substituted by the acceptor ACP. High-resolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre- and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA. PMID:27573844

  17. Partial deletion of beta9 loop in pancreatic lipase-related protein 2 reduces enzyme activity with a larger effect on long acyl chain substrates.

    PubMed

    Dridi, Kaouthar; Amara, Sawsan; Bezzine, Sofiane; Rodriguez, Jorge A; Carrière, Frédéric; Gaussier, Hélène

    2013-07-01

    Structural studies on pancreatic lipase have revealed a complex architecture of surface loops surrounding the enzyme active site and potentially involved in interactions with lipids. Two of them, the lid and beta loop, expose a large hydrophobic surface and are considered as acyl chain binding sites based on their interaction with an alkyl phosphonate inhibitor. While the role of the lid in substrate recognition and selectivity has been extensively studied, the implication of beta9 loop in acyl chain stabilization remained hypothetical. The characterization of an enzyme with a natural deletion of the lid, guinea pig pancreatic lipase-related protein 2 (GPLRP2), suggests however an essential contribution of the beta9 loop in the stabilization of the acyl enzyme intermediate formed during the lipolysis reaction. A GPLRP2 mutant with a seven-residue deletion of beta9 loop (GPLRP2-deltabeta9) was produced and its enzyme activity was measured using various substrates (triglycerides, monoglycerides, galactolipids, phospholipids, vinyl esters) with short, medium and long acyl chains. Whatever the substrate tested, GPLRP2-deltabeta9 activity is drastically reduced compared to that of wild-type GPLRP2 and this effect is more pronounced as the length of substrate acyl chain increases. Changes in relative substrate selectivity and stereoselectivity remained however weak. The deletion within beta9 loop has also a negative effect on the rate of enzyme inhibition by alkyl phosphonates. All these findings indicate that the reduced enzyme turnover observed with GPLRP2-deltabeta9 results from a weaker stabilization of the acyl enzyme intermediate due to a loss of hydrophobic interactions. PMID:24046870

  18. Enzyme Mechanism and Slow-Onset Inhibition of Plasmodium falciparum Enoyl-Acyl Carrier Protein Reductase by an Inorganic Complex

    PubMed Central

    de Medeiros, Patrícia Soares de Maria; Ducati, Rodrigo Gay; Basso, Luiz Augusto; Santos, Diógenes Santiago; da Silva, Luiz Hildebrando Pereira

    2011-01-01

    Malaria continues to be a major cause of children's morbidity and mortality worldwide, causing nearly one million deaths annually. The human malaria parasite, Plasmodium falciparum, synthesizes fatty acids employing the Type II fatty acid biosynthesis system (FAS II), unlike humans that rely on the Type I (FAS I) pathway. The FAS II system elongates acyl fatty acid precursors of the cell membrane in Plasmodium. Enoyl reductase (ENR) enzyme is a member of the FAS II system. Here we present steady-state kinetics, pre-steady-state kinetics, and equilibrium fluorescence spectroscopy data that allowed proposal of P. falciparum ENR (PfENR) enzyme mechanism. Moreover, building on previous results, the present study also evaluates the PfENR inhibition by the pentacyano(isoniazid)ferrateII compound. This inorganic complex represents a new class of lead compounds for the development of antimalarial agents focused on the inhibition of PfENR. PMID:21603269

  19. Enzyme Mechanism and Slow-Onset Inhibition of Plasmodium falciparum Enoyl-Acyl Carrier Protein Reductase by an Inorganic Complex.

    PubMed

    de Medeiros, Patrícia Soares de Maria; Ducati, Rodrigo Gay; Basso, Luiz Augusto; Santos, Diógenes Santiago; da Silva, Luiz Hildebrando Pereira

    2011-01-01

    Malaria continues to be a major cause of children's morbidity and mortality worldwide, causing nearly one million deaths annually. The human malaria parasite, Plasmodium falciparum, synthesizes fatty acids employing the Type II fatty acid biosynthesis system (FAS II), unlike humans that rely on the Type I (FAS I) pathway. The FAS II system elongates acyl fatty acid precursors of the cell membrane in Plasmodium. Enoyl reductase (ENR) enzyme is a member of the FAS II system. Here we present steady-state kinetics, pre-steady-state kinetics, and equilibrium fluorescence spectroscopy data that allowed proposal of P. falciparum ENR (PfENR) enzyme mechanism. Moreover, building on previous results, the present study also evaluates the PfENR inhibition by the pentacyano(isoniazid)ferrateII compound. This inorganic complex represents a new class of lead compounds for the development of antimalarial agents focused on the inhibition of PfENR.

  20. Crystal structure of Streptococcus pneumoniae acyl carrier protein synthase: an essential enzyme in bacterial fatty acid biosynthesis

    PubMed Central

    Chirgadze, Nickolay Y.; Briggs, Steven L.; McAllister, Kelly A.; Fischl, Anthony S.; Zhao, Genshi

    2000-01-01

    Acyl carrier protein synthase (AcpS) catalyzes the formation of holo-ACP, which mediates the essential transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and lipids in the cell. Thus, AcpS plays an important role in bacterial fatty acid and lipid biosynthesis, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structure of the Streptococcus pneumoniae AcpS and AcpS complexed with 3′5′-ADP, a product of AcpS, at 2.0 and 1.9 Å resolution, respectively. The crystal structure reveals an α/β fold and shows that AcpS assembles as a tightly packed functional trimer, with a non-crystallographic pseudo-symmetric 3-fold axis, which contains three active sites at the interface between protomers. Only two active sites are occupied by the ligand molecules. Although there is virtually no sequence similarity between the S.pneumoniae AcpS and the Bacillus subtilis Sfp transferase, a striking structural similarity between both enzymes was observed. These data provide a starting point for structure-based drug design efforts towards the identification of AcpS inhibitors with potent antibacterial activity. PMID:11032795

  1. Significance of reductive metabolism in human intestine and quantitative prediction of intestinal first-pass metabolism by cytosolic reductive enzymes.

    PubMed

    Nishimuta, Haruka; Nakagawa, Tetsuya; Nomura, Naruaki; Yabuki, Masashi

    2013-05-01

    The number of new drug candidates that are cleared via non-cytochrome P450 (P450) enzymes has increased. However, unlike oxidation by P450, the roles of reductive enzymes are less understood. The metabolism in intestine is especially not well known. The purposes of this study were to investigate the significance of reductive metabolism in human intestine, and to establish a quantitative prediction method of intestinal first-pass metabolism by cytosolic reductive enzymes, using haloperidol, mebendazole, and ziprasidone. First, we estimated the metabolic activities for these compounds in intestine and liver using subcellular fractions. Metabolic activities were detected in human intestinal cytosol (HIC) for all three compounds, and the intrinsic clearance values were higher than those in human liver cytosol for haloperidol and mebendazole. These metabolic activities in HIC were NADPH- and/or NADH-dependent. Furthermore, the metabolic activities for all three compounds in HIC were largely inhibited by menadione, which has been used as a carbonyl reductase (CBR)-selective chemical inhibitor. Therefore, considering subcellular location, cofactor requirement, and chemical inhibition, these compounds might be metabolized by CBRs in human intestine. Subsequently, we tried to quantitatively predict intestinal availability (F(g)) for these compounds using human intestinal S9 (HIS9). Our prediction model using apparent permeability of parallel artificial membrane permeability assay and metabolic activities in HIS9 could predict F(g) in humans for the three compounds well. In conclusion, CBRs might have higher metabolic activities in human intestine than in human liver. Furthermore, our prediction method of human F(g) using HIS9 is applicable to substrates of cytosolic reductive enzymes.

  2. Pancreatic digestive enzyme blockade in the intestine increases survival after experimental shock.

    PubMed

    DeLano, Frank A; Hoyt, David B; Schmid-Schönbein, Geert W

    2013-01-23

    Shock, sepsis, and multiorgan failure are associated with inflammation, morbidity, and high mortality. The underlying pathophysiological mechanism is unknown, but evidence suggests that pancreatic enzymes in the intestinal lumen autodigest the intestine and generate systemic inflammation. Blocking these enzymes in the intestine reduces inflammation and multiorgan dysfunction. We investigated whether enzymatic blockade also reduces mortality after shock. Three rat shock models were used here: hemorrhagic shock, peritonitis shock induced by placement of cecal material into the peritoneum, and endotoxin shock. One hour after initiation of hemorrhagic, peritonitis, or endotoxin shock, animals were administered one of three different pancreatic enzyme inhibitors--6-amidino-2-naphtyl p-guanidinobenzoate dimethanesulfate, tranexamic acid, or aprotinin--into the lumen of the small intestine. In all forms of shock, blockade of digestive proteases with protease inhibitor attenuated entry of digestive enzymes into the wall of the intestine and subsequent autodigestion and morphological damage to the intestine, lung, and heart. Animals treated with protease inhibitors also survived in larger numbers than untreated controls over a period of 12 weeks. Surviving animals recovered completely and returned to normal weight within 14 days after shock. The results suggest that the active and concentrated digestive enzymes in the lumen of the intestine play a central role in shock and multiorgan failure, which can be treated with protease inhibitors that are currently available for use in the clinic.

  3. Hydrogen-bonding in enzyme catalysis. Fourier-transform infrared detection of ground-state electronic strain in acyl-chymotrypsins and analysis of the kinetic consequences.

    PubMed Central

    White, A J; Wharton, C W

    1990-01-01

    I.r. difference spectra are presented for 3-(indol-3-yl)acryloyl-, cinnamoyl-, 3-(5-methylthien-2-yl)acryloyl-, dehydrocinnamoyl- and dihydrocinnamoyl-chymotrypsins at low pH, where the acyl-enzymes are catalytically inactive. At least two absorption bands are seen in each case in the ester carbonyl stretching region of the spectrum. Cinnamoyl-chymotrypsin substituted at the carbonyl carbon atom with 13C was prepared. A difference spectrum in which 13C-substituted acyl-enzyme was subtracted from [12C]acyl-enzyme shows two bands in the ester carbonyl region and thus confirms the assignment of the features to the single ester carbonyl group. The frequencies of the ester carbonyl bands are interpreted in terms of differential hydrogen-bonding. In each case a lower-frequency relatively narrow band is assigned to a productive potentially reactive binding mode in which the carbonyl oxygen atom is inserted in the oxyanion hole of the enzyme active centre. The higher-frequency band, which is broader, is assigned to a non-productive binding mode in each case, where a water molecule bridges from the carbonyl oxygen atom to His-57; this mode is equivalent to the crystallographically determined structure of 3-(indol-3-yl)acryloyl-chymotrypsin, i.e. the Henderson structure. A difference spectrum of dihydrocinnamoyl-chymotrypsin taken at higher pH shows resolution of a feature centred upon 1731 cm-1, which is assigned to a non-bonded conformer in which the carbonyl oxygen atom is not hydrogen-bonded. Perturbation of the protein spectrum in the presence of acyl groups is interpreted in terms of enhanced structural rigidity. It is reported that the ester carbonyl region of the difference spectrum of cinnamoyl-subtilisin is complicated by overlap of features that arise from protein perturbation. Measurements of carbonyl absorption frequencies in a number of solvents of the methyl esters of the acyl groups used to make acyl-enzymes have permitted determination of the apparent

  4. Intestine-specific Deletion of Acyl-CoA:Monoacylglycerol Acyltransferase (MGAT) 2 Protects Mice from Diet-induced Obesity and Glucose Intolerance*

    PubMed Central

    Nelson, David W.; Gao, Yu; Yen, Mei-I; Yen, Chi-Liang Eric

    2014-01-01

    The absorption of dietary fat involves the re-esterification of digested triacylglycerol in the enterocytes, a process catalyzed by acyl-CoA:monoacylglycerol acyltransferase (MGAT) 2. Mice without a functional gene encoding MGAT2 (Mogat2−/−) are protected from diet-induced obesity. Surprisingly, these mice absorb normal amounts of dietary fat but increase their energy expenditure. MGAT2 is expressed in tissues besides intestine, including adipose tissue in both mice and humans. To test the hypothesis that intestinal MGAT2 regulates systemic energy balance, we generated and characterized mice deficient in MGAT2 specifically in the small intestine (Mogat2IKO). We found that, like Mogat2−/− mice, Mogat2IKO mice also showed a delay in fat absorption, a decrease in food intake, and a propensity to use fatty acids as fuel when first exposed to a high fat diet. Mogat2IKO mice increased energy expenditure although to a lesser degree than Mogat2−/− mice and were protected against diet-induced weight gain and associated comorbidities, including hepatic steatosis, hypercholesterolemia, and glucose intolerance. These findings illustrate that intestinal lipid metabolism plays a crucial role in the regulation of systemic energy balance and may be a feasible intervention target. In addition, they suggest that MGAT activity in extraintestinal tissues may also modulate energy metabolism. PMID:24784138

  5. Intestine-specific deletion of acyl-CoA:monoacylglycerol acyltransferase (MGAT) 2 protects mice from diet-induced obesity and glucose intolerance.

    PubMed

    Nelson, David W; Gao, Yu; Yen, Mei-I; Yen, Chi-Liang Eric

    2014-06-20

    The absorption of dietary fat involves the re-esterification of digested triacylglycerol in the enterocytes, a process catalyzed by acyl-CoA:monoacylglycerol acyltransferase (MGAT) 2. Mice without a functional gene encoding MGAT2 (Mogat2(-/-)) are protected from diet-induced obesity. Surprisingly, these mice absorb normal amounts of dietary fat but increase their energy expenditure. MGAT2 is expressed in tissues besides intestine, including adipose tissue in both mice and humans. To test the hypothesis that intestinal MGAT2 regulates systemic energy balance, we generated and characterized mice deficient in MGAT2 specifically in the small intestine (Mogat2(IKO)). We found that, like Mogat2(-/-) mice, Mogat2(IKO) mice also showed a delay in fat absorption, a decrease in food intake, and a propensity to use fatty acids as fuel when first exposed to a high fat diet. Mogat2(IKO) mice increased energy expenditure although to a lesser degree than Mogat2(-/-) mice and were protected against diet-induced weight gain and associated comorbidities, including hepatic steatosis, hypercholesterolemia, and glucose intolerance. These findings illustrate that intestinal lipid metabolism plays a crucial role in the regulation of systemic energy balance and may be a feasible intervention target. In addition, they suggest that MGAT activity in extraintestinal tissues may also modulate energy metabolism.

  6. A comparative study on the metabolism of Epimedium koreanum Nakai-prenylated flavonoids in rats by an intestinal enzyme (lactase phlorizin hydrolase) and intestinal flora.

    PubMed

    Zhou, Jing; Chen, Yan; Wang, Ying; Gao, Xia; Qu, Ding; Liu, Congyan

    2013-12-24

    The aim of this study was to compare the significance of the intestinal hydrolysis of prenylated flavonoids in Herba Epimedii by an intestinal enzyme and flora. Flavonoids were incubated at 37 °C with rat intestinal enzyme and intestinal flora. HPLC-UV was used to calculate the metabolic rates of the parent drug in the incubation and LC/MS/MS was used to determine the chemical structures of metabolites generated by different flavonoid glycosides. Rates of flavonoid metabolism by rat intestinal enzyme were quicker than those of intestinal flora. The sequence of intestinal flora metabolic rates was icariin>epimedin B>epimedin A>epimedin C>baohuoside I, whereas the order of intestinal enzyme metabolic rates was icariin>epimedin A>epimedin C>epimedin B>baohuoside I. Meanwhile, the LC/MS/MS graphs showed that icariin produced three products, epimedin A/B/C had four and baohuoside I yielded one product in incubations of both intestinal enzyme and flora, which were more than the results of HPLC-UV due to the fact LC/MS/MS has lower detectability and higher sensitivity. Moreover, the outcomes indicated that the rate of metabolization of flavonoids by intestinal enzyme were faster than those of intestinal flora, which was consistent with the HPLC-UV results. In conclusion, the metabolic pathways of the same components by intestinal flora and enzyme were the same. What's more, an intestinal enzyme such as lactase phlorizin hydrolase exhibited a more significant metabolic role in prenylated flavonoids of Herba Epimedi compared with intestinal flora.

  7. An Investigation into the Gastrointestinal Stability of Exenatide in the Presence of Pure Enzymes, Everted Intestinal Rings and Intestinal Homogenates.

    PubMed

    Sun, Yanan; Wang, Mengshu; Sun, Bingxue; Li, Feng; Liu, Shubo; Zhang, Yong; Zhou, Yan; Chen, Yan; Kong, Wei

    2016-01-01

    The purpose of this study was to investigate the gastrointestinal stability of exenatide to determine the key factor(s) contributing to peptide degradation during the oral delivery process. The effects of pH and various digestive enzymes on the degradation kinetics of exenatide were determined. Moreover, the degradation clearances of peptide were also examined using rat everted intestinal rings and intestinal homogenates from various intestinal locations. Exenatide was comparatively stable within a pH range of 1.2-8. However, obvious degradation was observed in the presence of digestive enzymes. The order of enzymes, in terms of ability to degradate exenatide, was chymotrypsin>aminopeptidase N>carboxypeptidase A>trypsin>pepsin. Chymotrypsin showed the greatest ability to degrade exenatide (half-life t1/2, 5.784×10(-2) h), whereas aminopeptidase N and carboxylpeptidase A gave t1/2 values of 3.53 and 10.16 h, respectively. The degradation of exenatide was found to be peptide concentration- and intestinal site-dependent, with a lower clearance in the upper part of the duodenum and the lower part of the ileum. When using intestinal homogenates as enzyme sources, the order, in terms of peptide degradation ability, was ileum>jejunum>duodenum. However, no significant difference was observed in the remaining peptide concentrations throughout 2 h of incubation, which may be due to the involvement of cytosolic enzymes. These results revealed key factors contributing to peptide degradation, and suggest that the inhibition of chymotrypsin and site-specific delivery of exenatide might be advantageous in overcoming metabolic obstacles during its oral delivery.

  8. Generation of in vivo activating factors in the ischemic intestine by pancreatic enzymes

    NASA Astrophysics Data System (ADS)

    Mitsuoka, Hiroshi; Kistler, Erik B.; Schmid-Schönbein, Geert W.

    2000-02-01

    One of the early events in physiological shock is the generation of activators for leukocytes, endothelial cells, and other cells in the cardiovascular system. The mechanism by which these activators are produced has remained unresolved. We examine here the hypothesis that pancreatic digestive enzymes in the ischemic intestine may be involved in the generation of activators during intestinal ischemia. The lumen of the small intestine of rats was continuously perfused with saline containing a broadly acting pancreatic enzyme inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, 0.37 mM) before and during ischemia of the small intestine by splanchnic artery occlusion. This procedure inhibited activation of circulating leukocytes during occlusion and reperfusion. It also prevented the appearance of activators in portal venous and systemic artery plasma and attenuated initiating symptoms of multiple organ injury in shock. Intestinal tissue produces only low levels of activators in the absence of pancreatic enzymes, whereas in the presence of enzymes, activators are produced in a concentration- and time-dependent fashion. The results indicate that pancreatic digestive enzymes in the ischemic intestine serve as an important source for cell activation and inflammation, as well as multiple organ failure.

  9. Evolution of Acyl-Substrate Recognition by a Family of Acyl-Homoserine Lactone Synthases

    PubMed Central

    Christensen, Quin H.; Brecht, Ryan M.; Dudekula, Dastagiri; Greenberg, E. Peter; Nagarajan, Rajesh

    2014-01-01

    Members of the LuxI protein family catalyze synthesis of acyl-homoserine lactone (acyl-HSL) quorum sensing signals from S-adenosyl-L-methionine and an acyl thioester. Some LuxI family members prefer acyl-CoA, and others prefer acyl-acyl carrier protein (ACP) as the acyl-thioester substrate. We sought to understand the evolutionary history and mechanisms mediating this substrate preference. Our phylogenetic and motif analysis of the LuxI acyl-HSL synthase family indicates that the acyl-CoA-utilizing enzymes evolved from an acyl-ACP-utilizing ancestor. To further understand how acyl-ACPs and acyl-CoAs are recognized by acyl-HSL synthases we studied BmaI1, an octanoyl-ACP-dependent LuxI family member from Burkholderia mallei, and BjaI, an isovaleryl-CoA-dependent LuxI family member from Bradyrhizobium japonicum. We synthesized thioether analogs of their thioester acyl-substrates to probe recognition of the acyl-phosphopantetheine moiety common to both acyl-ACP and acyl-CoA substrates. The kinetics of catalysis and inhibition of these enzymes indicate that they recognize the acyl-phosphopantetheine moiety and they recognize non-preferred substrates with this moiety. We find that CoA substrate utilization arose through exaptation of acyl-phosphopantetheine recognition in this enzyme family. PMID:25401334

  10. The metabolic profile of acteoside produced by human or rat intestinal bacteria or intestinal enzyme in vitro employed UPLC-Q-TOF-MS.

    PubMed

    Cui, Qingling; Pan, Yingni; Xu, Xiaotong; Zhang, Wenjie; Wu, Xiao; Qu, Shouhe; Liu, Xiaoqiu

    2016-03-01

    Acteoside, the main and representative phenylethanoid glycosides of Herba Cistanches, possesses wide bioactivities but low oral bioavailability. It may serve as the prodrug and be converted into the active forms in gastrointestinal tract, which mainly occurred in intestinal tract composed of intestinal bacteria and intestinal enzyme. Intestinal bacteria, a new drug target, take a significant role on exerting pharmacological effects of drugs by oral administration. In this paper, acteoside was incubated with human or rat intestinal bacteria or rat intestinal enzyme for 36 h to seek metabolites responsible for pharmacodynamics. The samples were analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Besides the parent compound, 14 metabolites were detected and identified based on their retention times and fragmentation patterns in their MS spectra including 8 degradation metabolites, 2 isomers in intestinal bacteria and intestinal enzyme samples and 4 parent metabolites only found in intestinal enzymes. The metabolic pathway of acteoside was thus proposed. Identification of these metabolites of acteoside by the intestinal bacteria or intestinal enzyme gave an insight to clarify pharmacological mechanism of traditional Chinese medicines and identify the real active molecules.

  11. Genomic Analysis Reveals Versatile Organisms for Quorum Quenching Enzymes: Acyl-Homoserine Lactone-Acylase and -Lactonase

    PubMed Central

    Kalia, Vipin Chandra; Raju, Sajan C; Purohit, Hemant J

    2011-01-01

    Microbial virulence and their resistance to multiple drugs have obliged researchers to look for novel drug targets. Virulence of pathogenic microbes is regulated by signal molecules such as acylated homoserine lactone (AHL) produced during a cell density dependent phenomenon of quorum sensing (QS). In contrast, certain microbes produce AHL-lactonases and -acylases to degrade QS signals, also termed as quorum quenching. Mining sequenced genome databases has revealed organisms possessing conserved domains for AHL-lactonases and –acylases: i) Streptomyces (Actinobacteria), ii) Deinococcus (Deinococcus-Thermus), iii) Hyphomonas (α-Proteobacteria), iv) Ralstonia (β-Proteobacteria), v) Photorhabdus (γ-Proteobacteria), and certain marine gamma proteobacterium. Presence of genes for both the enzymes within an organism was observed in the following: i) Deinococcus radiodurans R1, ii) Hyphomonas neptunium ATCC 15444 and iii) Photorhabdus luminescens subsp. laumondii TTO1. These observations are supported by the presence motifs for lactonase and acylase in these strains. Phylogenetic analysis and multiple sequence alignment of the gene sequences for AHL-lactonases and –acylases have revealed consensus sequences which can be used to design primers for amplifying these genes even among mixed cultures and metagenomes. Quorum quenching can be exploited to prevent food spoilage, bacterial infections and bioremediation. PMID:21660112

  12. Histochemical distribution of intestinal enzymes of juvenile pacu (Piaractus mesopotamicus) fed lyophilized bovine colostrum.

    PubMed

    Moretti, Débora B; Nordi, Wiolene M; Cruz, Thaline M P; Cyrino, José Eurico P; Machado-Neto, Raul

    2014-10-01

    Enzyme activity was evaluated in the intestine of juvenile pacu, Piaractus mesopotamicus, fed diets containing 0, 10 or 20 % of lyophilized bovine colostrum (LBC) inclusion for either 30 or 60 days. The enzymes intestinal acid and alkaline phosphatase (ACP and ALP, respectively), nonspecific esterase (NSE), lipase (LIP), dipeptidyl aminopeptidase IV (DAP IV) and leucine aminopeptidase (LAP) were studied using histochemistry in four intestinal segments (S1, S2, S3 and rectum). Moderate activity of the DAP IV was detected in the three last intestinal segments, but no differences among the treatments were detected. Enzymes LAP, NSE and LIP were weakly stained in all intestinal segments and the inclusion of 10 or 20 % of LBC in the diet commanded a moderate reaction to NSE in the S3 segment at day 60. ACP activity was detected only in the brush border of the S1 segment of fish fed 0 % LBC for either 30 or 60 days. The activity of ALP was very strong in the first intestinal segment, but a weak reaction was seen in the last segments. The inclusion of 20 % of LBC changed the pattern of staining to the ALP, eliciting moderate staining in S2 at day 30 and S1 at day 60. The consumption of diets containing LBC by juvenile pacu did not have significant implications in intestinal enzymatic activity, which still was not fully stimulated. PMID:24823663

  13. Characterization and complete sequence of lactonase enzyme from Bacillus weihenstephanensis isolate P65 with potential activity against acyl homoserine lactone signal molecules.

    PubMed

    Sakr, Masarra Mohammed; Aboshanab, Khaled Mohamed Anwar; Aboulwafa, Mohammad Mabrouk; Hassouna, Nadia Abdel-Haleem

    2013-01-01

    Acyl homoserine lactones (AHLs) are the most common class of quorum sensing signal molecules (autoinducers) that have been reported to be essential for virulence of many relevant pathogenic bacteria such as Pseudomonas aeruginosa. New approach for controlling infections of such bacteria is through quorum quenching. In this study, the acyl homoserine lactone inhibitory activity of the crude enzyme from a Bacillus weihenstephanensis-isolate P65 was characterized. The crude enzyme was found to have relatively high thermal stability and was stable in pH range 6 to 9. The crude enzyme extract was found to have lactonase activity of 36.3 U/mg total protein. Maximum enzyme activity was achieved within a range of 28-50°C and pH 6-9. None of the metals used enhanced the activity neither did EDTA inhibit it. However, a concentration of 10 mM Fe(+2) reduced the activity to 73.8%. Catalytic activity and kinetic constants were determined using hexanoyl homoserine lactone as a substrate. Studying enzyme substrate specificity using synthetic standard signals displayed broad spectrum of activity. The enzyme was found to be constitutive. Isolation and complete nucleotide sequence of the respective lactonase gene were done and submitted to the Genbank database under accession code KC823046.

  14. Pancreatic digestive enzyme blockade in the small intestine prevents insulin resistance in hemorrhagic shock.

    PubMed

    DeLano, Frank A; Schmid-Schönbein, Geert W

    2014-01-01

    Hemorrhagic shock is associated with metabolic defects, including hyperglycemia and insulin resistance, but the mechanisms are unknown. We recently demonstrated that reduction of the extracellular domain of the insulin receptor by degrading proteases may lead to a reduced ability to maintain normal plasma glucose values. In shock, transfer of digestive enzymes from the lumen of the intestine into the systemic circulation after breakdown of the intestinal mucosal barrier causes inflammation and organ dysfunction. Suppression of the digestive enzymes in the lumen of the intestine with protease inhibitors is effective in reducing the level of the inflammatory reactions. To determine the degree to which blockade of digestive enzymes affects insulin resistance in shock, rats were exposed to acute hemorrhagic shock (mean arterial pressure of 30 mmHg for 2 h) at which time all shed blood volume was returned. Digestive proteases in the intestine were blocked with a serine protease inhibitor (tranexamic acid in polyethylene glycol and physiological electrolyte solution), and the density of the insulin receptor was measured with immunohistochemistry in the mesentery microcirculation. The untreated rat without enzyme blockade had significantly attenuated levels of insulin receptor density as compared with control and treated rats. Blockade of the digestive proteases after 60 min of hypotension in the lumen of the small intestine led to a lesser decrease in insulin receptor density compared with controls without protease blockade. Glucose tolerance test indicates a significant increase in plasma glucose levels 2 h after hemorrhagic shock, which are reduced to control values in the presence of protease inhibition in the lumen of the intestine. The transient reduction of the plasma glucose levels after an insulin bolus is significantly attenuated after shock but is restored when digestive enzymes in the lumen of the intestine are blocked. These results suggest that in

  15. 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

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

    PubMed Central

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

    2016-01-01

    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 isooctyl 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

  17. Novel C-9, 9'-O-acyl esters of (-)-carinol as free-radical scavengers and xanthine oxidase enzyme inhibitors: synthesis and biological evaluation.

    PubMed

    Suryadevara, Praveen Kumar; Tatipaka, Hari Babu; Vidadala, Rama Subba Rao; Tiwari, Ashok K; Rao, Janaswamy Madhusudana; Babu, Katragadda Suresh

    2013-02-01

    New compounds with hydrophyllic esters of (-)-carinol were synthesized and evaluated as xanthine oxidase enzyme inhibitors and antioxidants. Aliphatic esterfication of C-9,9'-OH groups of (-)-carinol resulted in lowering antioxidant and xanthine oxidase inhibitory activities. However certain aromatic acyl esters considerably improved the xathine oxidase inhibition. Aromatic esterification with electron withdrawing substitutions would preferred for improvement in XOD inhibition while retaining radical scavenging activity, electron withdrawing substitution led to the loss of free radical scavenging property and neutral substituents decrease the enzyme inhibitory potential.

  18. Examination of digestive enzyme distribution in gut tract and functions of intestinal caecum, in megascolecid earthworms (Oligochaeta: Megascolecidae) in Japan.

    PubMed

    Nozaki, Mana; Ito, Katsutoshi; Miura, Chiemi; Miura, Takeshi

    2013-09-01

    Earthworms ingest various materials in addition to food items, such as soil particles. Most earthworms of the family Megascolecidae, a dominant family in Japan, have intestinal caeca connected directly to the intestinal tract. The function of the caeca has not been demonstrated, although it is thought to be associated with digestion. We investigated the activity of the digestive enzymes amylase, phosphatase, cellulase, and protease in different regions of the gut, including the intestinal caeca, in three species of megascolecid earthworms, Pheretima heteropoda, Pheretima hilgendorfi, and Pheretima sieboldi. Activities of several enzymes were high in the intestinal caeca; in particular, protease activity was higher in the caeca than that in the anterior gut, foregut, midgut, and hindgut in all three species. Moreover, the ratio of enzyme activities in the intestinal caeca to whole-gut tended to be higher in manicate intestinal caeca than in simple intestinal caeca. These results suggest that the digestive system of earthworms relies on the intestinal caeca.

  19. ANTIOXIDANT ENZYME ACTIVITY AMONG ORPHANS INFECTED WITH INTESTINAL PARASITES IN PATHUM THANI PROVINCE, THAILAND.

    PubMed

    Mahittikorn, Aongart; Prasertbun, Rapeepan; Mori, Hirotake; Popruk, Supaluk

    2014-11-01

    Intestinal parasitic infections can negatively impact growth and nutrition in children. The infections can induce oxidative stress, resulting in a variety of illnesses. We measured antioxidant enzyme levels in orphan children infected with intestinal parasites to investigate the influence of nutritional status on antioxidant enzymes. This cross sectional study was conducted at an orphanage in Thailand. Stool samples were obtained from each subject and examined for intestinal parasites. Anthropometric measurements, complete blood count and biochemical parameters, including serum superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels, were obtained from studied subjects. One hundred twenty-eight children were included in the study. Intestinal parasites were found on microscopic examination of the stools in 36.7% (47/128); 18% (23/128) had a mixed parasite infection. Intestinal protozoa were found in 34.4% of subjects and intestinal helminthes were found in 2.3%. The median GPx level in children infected with intestinal parasites (2.3 ng/ml) was significantly lower than in non-infected children (7.7 ng/ml) (p < 0.05). However, there was no significant difference in SOD levels between the two groups. When comparing GPx levels in children with 1) pathogenic parasites, 2) non-pathogenic parasites and 3) no intestinal parasite infection, GPx levels differed significantly among three groups (2.2 ng/ml, 2.4 ng/ml and 7.7 ng/ml, respectively) (p < 0.05). When separating children by BMI and type of infection, the median SOD level in underweight children infected with pathogenic parasites (107.2 ng/ml) was significantly higher than in underweight children infected with non-pathogenic parasites (68.6 ng/ml) and without intestinal parasite infections (72.2 ng/ml). The present study identified two key findings: low GPx levels in children with intestinal parasitic infections, and the potential impact of malnutrition on some antioxidants. PMID:26466411

  20. Clarification on the decarboxylation mechanism in KasA based on the protonation state of key residues in the acyl-enzyme state.

    PubMed

    Lee, Wook; Engels, Bernd

    2013-07-11

    The β-ketoacyl ACP synthase I (KasA) is a promising drug target because it is essential for the survival of Mycobacterium tuberculosis , a causative agent of tuberculosis. It catalyzes a condensation reaction that comprises three steps. The resulting elongated acyl chains are subsequently needed for the cell wall construction. While the mechanism of the first step (acylation of Cys171 in the active site) is straightforward already, the second step (decarboxylation of malonyl substrate) has been controversial due to the difficulty in determining the correct protonation states of the involved residues (His311, His345, Lys340, Glu354). Available experimental data suggest three possible mechanisms which differ considerably. They are not consistent with each other because these studies could not be performed for KasA at the beginning of decarboxylation step (acyl-enzyme state of KasA). Instead, different mutants had to be used which are expected to resemble this situation. In this first computational study about this topic, we use the free energy perturbation (FEP) method to compute the relevant pKa values in the acyl-enzyme state of KasA and use molecular dynamics (MD) simulations to rationalize the results. Subsequent density functional theory (DFT)-based quantum mechanical/molecular mechanical (QM/MM) MD simulations and umbrella samplings have been used to disentangle the close relationships between the protonation states of the involved residues. By these simulations, we can address the preferred protonation states and roles of the residues involved in decarboxylation reaction, thereby suggesting the possible mechanism for the decarboxylation step. PMID:23768199

  1. Diagnosis of intestinal acariasis with avidin-biotin system enzyme-linked immunosorbent assay

    PubMed Central

    Zhang, Rong-Bo; Huang, Yong; Li, Chao-Pin; Cui, Yu-Bao

    2004-01-01

    AIM: To explore the value of avidin-biotin system enzyme-linked immunosorbent assay (ABC-ELISA) in diagnosis of intestinal acariasis. METHODS: Mite-specific IgG levels in serum of 48 patients with intestinal acariasis were measured with ABC-ELISA. The sensitivity of this method was compared with that of staphylococcal protein A enzyme-linked immunosorbent assay (SPA-ELISA). RESULTS: The positive rate of mite-specific IgG detected with ABC-ELISA and SPA-ELISA was 89.58% (43/48) and 56.25% (27/48), respectively. The positive rate with ABC-ELISA was statistically higher than that with SPA-ELISA (χ2 = 13.50, P < 0.01). CONCLUSION: ABC-ELISA is an effective method for the diagnosis of intestinal acariasis. PMID:15112362

  2. Seasonal effects on intestinal enzyme activity in the Australian agamid lizard, Lophognathus temporalis.

    PubMed

    Iglesias, Sebastian; Tracy, Christopher R; Bedford, Gavin S; McWhorter, Todd J; Christian, Keith A

    2009-05-01

    The tropical agamid lizard, Lophognathus temporalis, has higher metabolic and feeding rates during the wet season compared to the dry season. Also, lizards from urban sites tend to be larger than those from natural sites, partly due to site differences in food availability. Therefore, we hypothesized that activity of membrane-bound intestinal enzymes and masses of organs related to digestion would differ both seasonally and between urban and natural sites. To test this, we measured activities of aminopeptidase-N (APN), maltase, and sucrase, as well as organ masses. APN activity (micromol min(-1) g(-1)) was highest in the middle portion of the intestine (section 2), followed by the proximal portion (section 1) and then the distal portion (section 3). Maltase activity was highest in section 1 and decreased distally. We detected some sucrase activity in section 1 but none in sections 2 or 3. We found similar enzyme activities within each section irrespective of site or season. However, total enzyme activities were higher during the wet season compared to the dry season for both urban and bush L. temporalis. Total wet season enzyme activity in urban and bush L. temporalis was greatest for APN (25.4; 15.8 micromol min(-1); respectively), then maltase (3.9; 3.6 micromol min(-1); respectively) and then sucrase (0.3; 0.2 micromol min(-1); respectively). The higher total enzyme activities was the result of an increase in intestinal mass during the wet season.

  3. Acyl-acyl carrier protein: Lysomonogalactosyldiacylglycerol acyl transferase in Anabaena variabilis

    SciTech Connect

    Chen, H.H.

    1989-01-01

    Monogalactosyldiacylglycerol was produced when membranes isolated from the cyanobacterium, Anabaena variabilis, and washed free of soluble endogenous constituents, were incubated with ({sup 14}C)acyl-acyl carrier protein. This enzymatic synthesis of monogalactosyldiacylglycerol localized in the membranes was not dependent on any added cofactors, such as ATP, coenzyme A, and dithiothreitol. Palmitoyl-, stearoyl-, and oleoyl-acyl carrier proteins were approximately equally active as substrates with Km of 0.37, 0.36, and 0.23 {mu}M, respectively. The ({sup 14}C)acyl group was exclusively transferred to the sn-1 hydroxyl of the glycerol backbone of monogalactosyldiacylglycerol as demonstrated by hydrolysis of all incorporated acyl groups by the lipase from Rhizopus arrhizus delamar. Using a double labelled ({sup 14}C)acyl-({sup 14}C)acyl carrier protein, this enzyme catalyzed the direct transfer of the acyl group from acyl-acyl carrier protein to an endogenous lysomonogalactosyldiacylglycerol to form monogalactosyldiacylglycerol. The transfer reaction mechanism was also confirmed by the increased activity with the addition of the lysomonogalactosyldiacylglycerol suspension. A specific galactolipid acyl hydrolase activity was released into the soluble protein fraction when the membranes of Anabaena variabilis were treated with 2% Triton X-100. The positional specificity of this acyl hydrolase was demonstrated to be similar to that of Rhizopus lipase, i.e. only the acyl group at the sn-1 position was hydrolyzed. The acyl hydrolase which was also localized in the membrane fraction of Anabaena variabilis was presumably responsible for producing endogenous lysomonogalactosyldiacylglycerol used by the acyltransferase.

  4. The Secretion and Action of Brush Border Enzymes in the Mammalian Small Intestine.

    PubMed

    Hooton, Diane; Lentle, Roger; Monro, John; Wickham, Martin; Simpson, Robert

    2015-01-01

    Microvilli are conventionally regarded as an extension of the small intestinal absorptive surface, but they are also, as latterly discovered, a launching pad for brush border digestive enzymes. Recent work has demonstrated that motor elements of the microvillus cytoskeleton operate to displace the apical membrane toward the apex of the microvillus, where it vesiculates and is shed into the periapical space. Catalytically active brush border digestive enzymes remain incorporated within the membranes of these vesicles, which shifts the site of BB digestion from the surface of the enterocyte to the periapical space. This process enables nutrient hydrolysis to occur adjacent to the membrane in a pre-absorptive step. The characterization of BB digestive enzymes is influenced by the way in which these enzymes are anchored to the apical membranes of microvilli, their subsequent shedding in membrane vesicles, and their differing susceptibilities to cleavage from the component membranes. In addition, the presence of active intracellular components of these enzymes complicates their quantitative assay and the elucidation of their dynamics. This review summarizes the ontogeny and regulation of BB digestive enzymes and what is known of their kinetics and their action in the peripheral and axial regions of the small intestinal lumen.

  5. 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.

  6. Acylated Carrageenan Changes the Physicochemical Properties of Mixed Enzyme-Lipid Ultrathin Films and Enhances the Catalytic Properties of Sucrose Phosphorylase Nanostructured as Smart Surfaces.

    PubMed

    Rocha, Jefferson M; Pavinatto, Adriana; Nobre, Thatyane M; Caseli, Luciano

    2016-06-23

    Control over the catalytic activity of enzymes is important to construct biosensors with a wide range of detectability and higher stability. For this, immobilization of enzymes on solid supports as nanostructured films is a current approach that permits easy control of the molecular architecture as well as tuning of the properties. In this article, we employed acylated carrageenan (AC) mixed with phospholipids at the air-water interface to facilitate the adsorption of the enzyme sucrose phosphorylase (SP). AC stabilized the adsorption of SP at the phospholipid monolayer, as detected by tensiometry, by which thermodynamic parameters could be inferred from the surface pressure-area isotherm. Also, infrared spectroscopy applied in situ over the monolayer showed that the AC-phospholipid system not only permitted the enzyme to be adsorbed but also helped conserve its secondary structure. The mixed monolayers were then transferred onto solid supports as Langmuir-Blodgett (LB) films and investigated with transfer ratio, quartz crystal microbalance, fluorescence spectroscopy, and atomic force microscopy. The enzyme activity of the LB film was then determined, revealing that although there was an expected reduction in activity in relation to the homogeneous environment the activity could be better preserved after 1 month, revealing enhanced stability. PMID:27249064

  7. Digestive enzyme expression and epithelial structure of small intestine in neonatal rats after 16 days spaceflight

    NASA Astrophysics Data System (ADS)

    Miyake, M.; Yamasaki, M.; Hazama, A.; Ijiri, K.; Shimizu, T.

    It is important to assure whether digestive system can develop normally in neonates during spaceflight. Because the small intestine changes its function and structure drastically around weaning known as redifferentiation. Lactase expression declines and sucrase increases in small intestine for digestion of solid food before weaning. In this paper, we compared this enzyme transition and structural development of small intestine in neonatal rats after spaceflight. To find digestive genes differentially expressed in fight rats, DNA membrane macroarray was also used. Eight-day old rats were loaded to Space Shuttle Columbia, and housed in the animal facility for 16 days in space (STS-90, Neurolab mission). Two control groups (AGC; asynchronous ground control and VIV; vivarium) against flight group (FLT) were prepared. There was no difference in structure (crypt depth) and cell differentiation of epithelium between FLT and AGC by immunohistochemical analysis. We found that the amount of sucrase mRNA compared to lactase was decreased in FLT by RT-PCR. It reflected the enzyme transition was inhibited. Increase of 5 genes (APO A-I, APO A-IV, ACE, aFABP and aminopeptidase M) and decrease of carboxypeptidase-D were detected in FLT using macroarray. We think nutrition differences (less nourishment and late weaning) during spaceflight may cause inhibition of enzyme transition at least partly. The weightlessness might contribute to the inhibition through behavioral change.

  8. 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.

  9. SIRT3 and SIRT5 regulate the enzyme activity and cardiolipin binding of very long-chain acyl-CoA dehydrogenase.

    PubMed

    Zhang, Yuxun; Bharathi, Sivakama S; Rardin, Matthew J; Uppala, Radha; Verdin, Eric; Gibson, Bradford W; Goetzman, Eric S

    2015-01-01

    SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane. PMID:25811481

  10. Enzyme activities of intestinal triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.).

    PubMed

    Oxley, Anthony; Torstensen, Bente E; Rustan, Arild C; Olsen, Rolf E

    2005-05-01

    The substitution of fish oil with plant-derived oil in diets for carnivorous fish, such as Atlantic salmon, has previously revealed the potentially deleterious supranuclear accumulation of lipid droplets in intestinal cells (enterocytes) which may compromise gut integrity, and consequently, fish health. This suggests that unfamiliar dietary lipid sources may have a significant impact on intestinal lipid metabolism, however, the mode of lipid resynthesis is largely unknown in teleost fish intestine. The present study aimed at characterising three key lipogenic enzymes involved in the biosynthesis of triacylglycerol (TAG) and phosphatidylcholine (PC) in Atlantic salmon enterocytes: monoacylglycerol acyltransferase (MGAT), diacylglycerol acyltransferase (DGAT), and diacylglycerol cholinephosphotransferase (CPT). Furthermore, to investigate the dietary effect of plant oils on these enzymes, two experimental groups of fish were fed a diet with either capelin (fish oil) or vegetable oil (rapeseed oil:palm oil:linseed oil, 55:30:15 w/w) as the lipid source. The monoacylglycerol (MAG) pathway was highly active in the intestinal mucosa of Atlantic salmon as demonstrated by MGAT activity (7 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)) and DGAT activity (4 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)), with MGAT appearing to also provide adequate production of sn-1,2-diacylglycerol for potential utilisation in PC synthesis via CPT activity (0.4 nmol CDP-[(14)C]choline incorporated min(-1) mg protein(-1)). Both DGAT and CPT specific activity values were comparable to reported mammalian equivalents, although MGAT activity was lower. Nevertheless, MGAT appeared not to be the rate-limiting step in salmon intestinal TAG synthesis. The homology between piscine and mammalian enzymes was established by similar stimulation and inhibition profiles by a variety of tested cofactors and isomeric substrates. The low dietary n-3/n-6 PUFA ratio

  11. Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells.

    PubMed

    Ringqvist, Emma; Palm, J E Daniel; Skarin, Hanna; Hehl, Adrian B; Weiland, Malin; Davids, Barbara J; Reiner, David S; Griffiths, William J; Eckmann, Lars; Gillin, Frances D; Svärd, Staffan G

    2008-06-01

    Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell-cell interactions, since only small amounts of arginine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine. PMID:18359106

  12. Intestine.

    PubMed

    Smith, J M; Skeans, M A; Horslen, S P; Edwards, E B; Harper, A M; Snyder, J J; Israni, A K; Kasiske, B L

    2016-01-01

    Intestine and intestine-liver transplant plays an important role in the treatment of intestinal failure, despite decreased morbidity associated with parenteral nutrition. In 2014, 210 new patients were added to the intestine transplant waiting list. Among prevalent patients on the list at the end of 2014, 65% were waiting for an intestine transplant and 35% were waiting for an intestine-liver transplant. The pretransplant mortality rate decreased dramatically over time for all age groups. Pretransplant mortality was highest for adult candidates, at 22.1 per 100 waitlist years compared with less than 3 per 100 waitlist years for pediatric candidates, and notably higher for candidates for intestine-liver transplant than for candidates for intestine transplant without a liver. Numbers of intestine transplants without a liver increased from a low of 51 in 2013 to 67 in 2014. Intestine-liver transplants increased from a low of 44 in 2012 to 72 in 2014. Short-gut syndrome (congenital and other) was the main cause of disease leading to both intestine and intestine-liver transplant. Graft survival improved over the past decade. Patient survival was lowest for adult intestine-liver recipients and highest for pediatric intestine recipients.

  13. Subacute effects of carbofuran on enzyme functions in rat small intestine.

    PubMed

    Gera, Nidhi; Kiran, Ravi; Mahmood, Akhtar

    2009-02-01

    The effect of carbofuran administration to rats has been studied on enzymes functions in rat intestine. Carbofuran was administrated 4.0 mg/kg body weight for 7 days or 2.8 mg/kg body weight for 30 days daily by Ryle's tube. Animals given carbofuran for 30 days exhibited retarded growth compared to control group. The activities of sucrase (56%), alkaline phosphatase (62%), leucine aminopeptidase (56%), and gamma-glutamyl trans peptidase (84%) were enhanced in animals given carbofuran for 7 days. Enhancement in the activities of alkaline phosphatase and leucine amino peptidase (92-96%) was also observed in animals exposed to carbofuran for 30 days, but the activities of sucrase (28%) and gamma-glutamyl transpeptidase (49%) were reduced under these conditions. There was no change in activities of maltase, lactase, and trehalase in pesticide-treated animals for 7 or 30 days. The activity of lactate dehydrogenase was enhanced (p < 0.001) in 7 days and 30 days induced carbofuran toxicity. The activities of glucose-6-phosphatase and glutamate pyruvate transaminase were also enhanced (p < 0.001) in pesticide-treated animals for 7 days, but were reduced by 46% and 26%, respectively, after 30 days of carbofuran exposure. The activity of glutamate oxaloacetate transaminase was unaltered in carbofuran toxicity. Kinetic analysis of brush border enzymes revealed a change in V(max) with no change in apparent Km. Western blot analysis of brush border sucrase, alkaline phosphatase, and leucine aminopeptidase corroborated the enzyme activity data. Intestinal histological revealed distruption of the villi, and comet assay showed disintegration of DNA in enterocytes of animals exposed to carbofuran for 30 days. These findings suggest that carbofuran toxicity may modulate digestive functions in rat intestine.

  14. Monogalactosyldiacylglycerol biosynthesis by direct acyl transfer in Anabaene variabilis

    SciTech Connect

    Chen, H.H.; Wickrema, A.; Jaworski, J.

    1987-04-01

    The authors previously reported the direct acylation of monogalactosyldiacylglycerol (MGDG) by an enzyme in the membranes of the cyanobacterium Anabaena variabilis. The enzyme requires acyl-acyl carrier protein (acyl-ACP) as substrate, but had no other additional cofactor requirements. Palmitoyl-, stearoyl- and oleoyl-ACP were all effective substrates. The A. variabilis membranes also had a hydrolase activity which metabolized the acyl-ACP to yield free fatty acid and ACP. Possible mechanisms for the acylation reaction include either acyl exchange with existing MGDG or direct acyl transfer to a lyso-MGDG, with concomitant release of free ACP. The mechanism of this reaction has been resolved using a double labelled (/sup 14/C)acyl-(/sup 14/)ACP substrate prepared with E. coli acyl-ACP synthetase. Following incubation with the enzyme, the unreacted (/sup 14/)acyl-(/sup 14/)ACP was isolated and the (/sup 14/)acyl/(/sup 14/)ACP ratio determined. Comparison of this ratio to that of the original substrate indicated no change and eliminated acyl exchange as a possible mechanism. Therefore, the direct acylation of lyso-MGDG is the proposed mechanism for this enzyme.

  15. 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

  16. 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

  17. Acyl-lipid metabolism.

    PubMed

    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

  18. Effects of xylitol on carbohydrate digesting enzymes activity, intestinal glucose absorption and muscle glucose uptake: a multi-mode study.

    PubMed

    Chukwuma, Chika Ifeanyi; Islam, Md Shahidul

    2015-03-01

    The present study investigated the possible mechanism(s) behind the effects of xylitol on carbohydrate digesting enzymes activity, muscle glucose uptake and intestinal glucose absorption using in vitro, ex vivo and in vivo experimental models. The effects of increasing concentrations of xylitol (2.5%-40% or 164.31 mM-2628.99 mM) on alpha amylase and alpha glucosidase activity in vitro and intestinal glucose absorption and muscle glucose uptake were investigated under ex vivo conditions. Additionally, the effects of an oral bolus dose of xylitol (1 g per kg BW) on gastric emptying and intestinal glucose absorption and digesta transit in the different segments of the intestinal tract were investigated in normal and type 2 diabetic rats at 1 hour after dose administration, when phenol red was used as a recovery marker. Xylitol exhibited concentration-dependent inhibition of alpha amylase (IC₅₀ = 1364.04 mM) and alpha glucosidase (IC₅₀ = 1127.52 mM) activity in vitro and small intestinal glucose absorption under ex vivo condition. Xylitol also increased dose dependent muscle glucose uptake with and without insulin, although the uptake was not significantly affected by the addition of insulin. Oral single bolus dose of xylitol significantly delayed gastric emptying, inhibited intestinal glucose absorption but increased the intestinal digesta transit rate in both normal and diabetic rats compared to their respective controls. The data of this study suggest that xylitol reduces intestinal glucose absorption via inhibiting major carbohydrate digesting enzymes, slowing gastric emptying and fastening the intestinal transit rate, but increases muscle glucose uptake in normal and type 2 diabetic rats.

  19. Influence of bacterial N-acyl-homoserine lactones on growth parameters, pigments, antioxidative capacities and the xenobiotic phase II detoxification enzymes in barley and yam bean.

    PubMed

    Götz-Rösch, Christine; Sieper, Tina; Fekete, Agnes; Schmitt-Kopplin, Philippe; Hartmann, Anton; Schröder, Peter

    2015-01-01

    Bacteria are able to communicate with each other and sense their environment in a population density dependent mechanism known as quorum sensing (QS). N-acyl-homoserine lactones (AHLs) are the QS signaling compounds of Gram-negative bacteria which are frequent colonizers of rhizospheres. While cross-kingdom signaling and AHL-dependent gene expression in plants has been confirmed, the responses of enzyme activities in the eukaryotic host upon AHLs are unknown. Since AHL are thought to be used as so-called plant boosters or strengthening agents, which might change their resistance toward radiation and/or xenobiotic stress, we have examined the plants' pigment status and their antioxidative and detoxifying capacities upon AHL treatment. Because the yield of a crop plant should not be negatively influenced, we have also checked for growth and root parameters. We investigated the influence of three different AHLs, namely N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL), and N-decanoyl- homoserine lactone (C10-HSL) on two agricultural crop plants. The AHL-effects on Hordeum vulgare (L.) as an example of a monocotyledonous crop and on the tropical leguminous crop plant Pachyrhizus erosus (L.) were compared. While plant growth and pigment contents in both plants showed only small responses to the applied AHLs, AHL treatment triggered tissue- and compound-specific changes in the activity of important detoxification enzymes. The activity of dehydroascorbate reductase in barley shoots after C10-HSL treatment for instance increased up to 384% of control plant levels, whereas superoxide dismutase activity in barley roots was decreased down to 23% of control levels upon C6-HSL treatment. Other detoxification enzymes reacted similarly within this range, with interesting clusters of positive or negative answers toward AHL treatment. In general the changes on the enzyme level were more severe in barley than in yam bean which might be due to the different abilities of the plants to

  20. Influence of bacterial N-acyl-homoserine lactones on growth parameters, pigments, antioxidative capacities and the xenobiotic phase II detoxification enzymes in barley and yam bean

    PubMed Central

    Götz-Rösch, Christine; Sieper, Tina; Fekete, Agnes; Schmitt-Kopplin, Philippe; Hartmann, Anton; Schröder, Peter

    2015-01-01

    Bacteria are able to communicate with each other and sense their environment in a population density dependent mechanism known as quorum sensing (QS). N-acyl-homoserine lactones (AHLs) are the QS signaling compounds of Gram-negative bacteria which are frequent colonizers of rhizospheres. While cross-kingdom signaling and AHL-dependent gene expression in plants has been confirmed, the responses of enzyme activities in the eukaryotic host upon AHLs are unknown. Since AHL are thought to be used as so-called plant boosters or strengthening agents, which might change their resistance toward radiation and/or xenobiotic stress, we have examined the plants’ pigment status and their antioxidative and detoxifying capacities upon AHL treatment. Because the yield of a crop plant should not be negatively influenced, we have also checked for growth and root parameters. We investigated the influence of three different AHLs, namely N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL), and N-decanoyl- homoserine lactone (C10-HSL) on two agricultural crop plants. The AHL-effects on Hordeum vulgare (L.) as an example of a monocotyledonous crop and on the tropical leguminous crop plant Pachyrhizus erosus (L.) were compared. While plant growth and pigment contents in both plants showed only small responses to the applied AHLs, AHL treatment triggered tissue- and compound-specific changes in the activity of important detoxification enzymes. The activity of dehydroascorbate reductase in barley shoots after C10-HSL treatment for instance increased up to 384% of control plant levels, whereas superoxide dismutase activity in barley roots was decreased down to 23% of control levels upon C6-HSL treatment. Other detoxification enzymes reacted similarly within this range, with interesting clusters of positive or negative answers toward AHL treatment. In general the changes on the enzyme level were more severe in barley than in yam bean which might be due to the different abilities of the plants to

  1. Developmental pattern of rat intestinal brush-border enzymic proteins along the villus--crypt axis.

    PubMed

    Simon, P M; Kedinger, M; Raul, F; Grenier, J F; Haffen, K

    1979-02-15

    At various postnatal stages, intestinal epithelial cells were isolated sequentially from villus tip to crypt base by successive EDTA treatments. According to the localization of marker enzymic activities, isolated cells were pooled into three cell compartments: villus (V), lower villus and upper crypt (VC) and crypt (C). Purified brush-border-membrane proteins were separated by 7.5%-polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Enzymic activities could be assigned to some protein bands: maltase/glucoamylase (protein band 3), sucrase-isomaltase (protein bands 3 and 6), lactase (protein band 5) and alkaline phosphatase (region of protein bands 8 and 9). The findings suggest the following. (1) Sucrase-isomaltase activities appeared in compartment C at 17 days with a simultaneous increase of the pre-existing protein band 3 and appearance of a well-defined protein band in position 6; the enzymic complex remained still present in the crypt cells until adulthood. From the day 21 onwards, sucrase-isomaltase was detected in compartments VC and V. (2) Lactase was only present in the three cell compartments until day 21; at this developmental stage its activity completely disappeared from compartment C, in spite of the persistence of a weak protein band. (3) Alkaline phosphatase activity could be detected as a single peak corresponding to protein band 9 in all three cell compartments until day 21; thereafter it was replaced by two peaks of activity showing a less precise correlation with the well-defined protein bands 8 and 9. In the crypt cells of the adult rat, however, the preweaning situation, which was regularly observed, is an unexpected phenomenon. (4) Maltase and glucoamylase did not display any marked qualitative or quantitative modifications either along the villus-crypt axis or during the period of postnatal development studied. Evidence is given from the present data that each brush-border enzyme investigated has a specific

  2. Intestinal triacylglycerol synthesis in fat absorption and systemic energy metabolism.

    PubMed

    Yen, Chi-Liang Eric; Nelson, David W; Yen, Mei-I

    2015-03-01

    The intestine plays a prominent role in the biosynthesis of triacylglycerol (triglyceride; TAG). Digested dietary TAG is repackaged in the intestine to form the hydrophobic core of chylomicrons, which deliver metabolic fuels, essential fatty acids, and other lipid-soluble nutrients to the peripheral tissues. By controlling the flux of dietary fat into the circulation, intestinal TAG synthesis can greatly impact systemic metabolism. Genes encoding many of the enzymes involved in TAG synthesis have been identified. Among TAG synthesis enzymes, acyl-CoA:monoacylglycerol acyltransferase 2 and acyl-CoA:diacylglycerol acyltransferase (DGAT)1 are highly expressed in the intestine. Their physiological functions have been examined in the context of whole organisms using genetically engineered mice and, in the case of DGAT1, specific inhibitors. An emerging theme from recent findings is that limiting the rate of TAG synthesis in the intestine can modulate gut hormone secretion, lipid metabolism, and systemic energy balance. The underlying mechanisms and their implications for humans are yet to be explored. Pharmacological inhibition of TAG hydrolysis in the intestinal lumen has been employed to combat obesity and associated disorders with modest efficacy and unwanted side effects. The therapeutic potential of inhibiting specific enzymes involved in intestinal TAG synthesis warrants further investigation. PMID:25231105

  3. Intestinal triacylglycerol synthesis in fat absorption and systemic energy metabolism

    PubMed Central

    Yen, Chi-Liang Eric; Nelson, David W.; Yen, Mei-I

    2015-01-01

    The intestine plays a prominent role in the biosynthesis of triacylglycerol (triglyceride; TAG). Digested dietary TAG is repackaged in the intestine to form the hydrophobic core of chylomicrons, which deliver metabolic fuels, essential fatty acids, and other lipid-soluble nutrients to the peripheral tissues. By controlling the flux of dietary fat into the circulation, intestinal TAG synthesis can greatly impact systemic metabolism. Genes encoding many of the enzymes involved in TAG synthesis have been identified. Among TAG synthesis enzymes, acyl-CoA:monoacylglycerol acyltransferase 2 and acyl-CoA:diacylglycerol acyltransferase (DGAT)1 are highly expressed in the intestine. Their physiological functions have been examined in the context of whole organisms using genetically engineered mice and, in the case of DGAT1, specific inhibitors. An emerging theme from recent findings is that limiting the rate of TAG synthesis in the intestine can modulate gut hormone secretion, lipid metabolism, and systemic energy balance. The underlying mechanisms and their implications for humans are yet to be explored. Pharmacological inhibition of TAG hydrolysis in the intestinal lumen has been employed to combat obesity and associated disorders with modest efficacy and unwanted side effects. The therapeutic potential of inhibiting specific enzymes involved in intestinal TAG synthesis warrants further investigation. PMID:25231105

  4. Intestinal triacylglycerol synthesis in fat absorption and systemic energy metabolism.

    PubMed

    Yen, Chi-Liang Eric; Nelson, David W; Yen, Mei-I

    2015-03-01

    The intestine plays a prominent role in the biosynthesis of triacylglycerol (triglyceride; TAG). Digested dietary TAG is repackaged in the intestine to form the hydrophobic core of chylomicrons, which deliver metabolic fuels, essential fatty acids, and other lipid-soluble nutrients to the peripheral tissues. By controlling the flux of dietary fat into the circulation, intestinal TAG synthesis can greatly impact systemic metabolism. Genes encoding many of the enzymes involved in TAG synthesis have been identified. Among TAG synthesis enzymes, acyl-CoA:monoacylglycerol acyltransferase 2 and acyl-CoA:diacylglycerol acyltransferase (DGAT)1 are highly expressed in the intestine. Their physiological functions have been examined in the context of whole organisms using genetically engineered mice and, in the case of DGAT1, specific inhibitors. An emerging theme from recent findings is that limiting the rate of TAG synthesis in the intestine can modulate gut hormone secretion, lipid metabolism, and systemic energy balance. The underlying mechanisms and their implications for humans are yet to be explored. Pharmacological inhibition of TAG hydrolysis in the intestinal lumen has been employed to combat obesity and associated disorders with modest efficacy and unwanted side effects. The therapeutic potential of inhibiting specific enzymes involved in intestinal TAG synthesis warrants further investigation.

  5. Expression of an antimicrobial peptide, digestive enzymes and nutrient transporters in the intestine of E. praecox-infected chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Coccidiosis is a major intestinal disease of poultry, caused by several species of the protozoan Eimeria. The objective of this study was to examine changes in expression of digestive enzymes, nutrient transporters and an antimicrobial peptide following an Eimeria praecox challenge of chickens at d...

  6. Stearoyl-Acyl Carrier Protein and Unusual Acyl-Acyl Carrier Protein Desaturase Activities Are Differentially Influenced by Ferredoxin1

    PubMed Central

    Schultz, David J.; Suh, Mi Chung; Ohlrogge, John B.

    2000-01-01

    Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Δ9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [14C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium × hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Δ9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction. PMID:11027717

  7. Effects of dietary stachyose on growth performance, digestive enzyme activities and intestinal morphology of juvenile turbot ( Scophthalmus maximus L)

    NASA Astrophysics Data System (ADS)

    Hu, Haibin; Zhang, Yanjiao; Mai, Kangsen; Ai, Qinghui; Xu, Wei; Zhang, Wenbing; Li, Yanxian; Liu, Jintao

    2015-10-01

    A 12-week feeding trial was conducted to evaluate the effects of dietary stachyose on the growth performance, digestive enzymes activities and intestinal structures of juvenile turbot ( Scophthalmus maximus L). Five isonitrogenous (49.58% crude protein) and isolipidic (10.50% crude lipid) diets were formulated to contain 0 (Control), 0.625% (S-0.625), 1.25% (S-1.25), 2.5% (S-2.5) and 5% (S-5) stachyose, respectively. With the increase of stachyose level, the growth performance and feed utilization of turbot, such as the specific growth rate, final mean body weight, weight gain rate and feed efficiency, increased significantly ( P< 0.05) and then stabilized. The feed intake of fish fed S-5 was significantly higher ( P< 0.05) than that of fish in other groups. The activities of trypsin, intestinal caseinolytic, stomach and intestinal amylase were significantly influenced by stachyose ( P<0.05). The highest values of trypsin and intestinal caseinolytic activities were observed in group S-1.25, while the highest activity of stomach amylase and the lowest activity of intestine amylase were observed in group S-5. No lesion or damage was found on the distal intestine structures of fish from all treatments, while the height of simple folds in the distal intestine was significantly increased ( P< 0.05) when 1.25% or 2.5% stachyose was added in the diets. These results indicated that moderate level of stachyose (1.25%) improves the growth performance, feed utilization, digestive enzyme activities and the distal intestine structures of juvenile turbot.

  8. 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

  9. Changes in messenger RNA of pancreatic enzymes and intestinal cholecystokinin after a 7-day bile-pancreatic juice diversion from the proximal small intestine in rats.

    PubMed

    Hara, H; Ochi, Y; Kasai, T

    1997-06-01

    We have previously demonstrated the bile-pancreatic juice (BPJ)-independent stimulation of pancreatic enzyme secretion in chronic BPJ-diverted rats. Pancreatic and intestinal adaptation to 7-day BPJ diversion was next examined. Pancreatic enzyme mRNA and cholecystokinin mRNA in the jejunal mucosa were measured in rats with BPJ diverted into the ileum (PBD rats) in comparison with the figures for rats with BPJ returned to the duodenum (normal rats) or laparotomized (Intact) rats under well-nourished conditions. Amylase mRNA in the pancreas was lower and trypsinogen plus chymotrypsinogen mRNA was higher in the PBD rats than in the intact rats. The change in pancreatic mRNA was similar to that in the specific activities of the enzymes after a chronic BPJ diversion. This finding suggests that these pancreatic enzymes were regulated by the mRNA level. The portal concentration of cholecystokinin in the postabsorptive period (exogenously non-stimulated status) was 4-fold higher in the PBD group than in the normal and intact groups. Cholecystokinin mRNA in the jejunal mucosa of PBD rats was somewhat higher than that of intact rats. These results suggest that intestinal cholecystokinin was predominantly increased at the translational or later stage by chronic BPJ diversion.

  10. Dynamic Evolution of the LPS-Detoxifying Enzyme Intestinal Alkaline Phosphatase in Zebrafish and Other Vertebrates

    PubMed Central

    Yang, Ye; Wandler, Anica M.; Postlethwait, John H.; Guillemin, Karen

    2012-01-01

    Alkaline phosphatases (Alps) are well-studied enzymes that remove phosphates from a variety of substrates. Alps function in diverse biological processes, including modulating host-bacterial interactions by dephosphorylating the Gram-negative bacterial cell wall component lipopolysaccharide (LPS). In animals, Alps are encoded by multiple genes characterized by either ubiquitous expression (named Alpls for their liver expression, but a key to proper bone mineralization), or their tissue-specific expression, for example in the intestine (Alpi). We previously characterized a zebrafish alpi gene (renamed here alpi.1) that is regulated by Myd88-dependent innate immune signaling and that is required to prevent a host’s excessive inflammatory reactions to its resident microbiota. Here we report the characterization of two new alp genes in zebrafish, alpi.2 and alp3. To understand their origins, we investigated the phylogenetic history of Alp genes in animals. We find that vertebrate Alp genes are organized in three clades with one of these clades missing from the mammals. We present evidence that these three clades originated during the two vertebrate genome duplications. We show that alpl is ubiquitously expressed in zebrafish, as it is in mammals, whereas the other three alps are specific to the intestine. Our phylogenetic analysis reveals that in contrast to Alpl, which has been stably maintained as a single gene throughout the vertebrates, the Alpis have been lost and duplicated multiple times independently in vertebrate lineages, likely reflecting the rapid and dynamic evolution of vertebrate gut morphologies, driven by changes in bacterial associations and diet. PMID:23091474

  11. Intestinal function and gut microflora of broiler chickens as influenced by cereal grains and microbial enzyme supplementation.

    PubMed

    Shakouri, M D; Iji, P A; Mikkelsen, L L; Cowieson, A J

    2009-10-01

    A study was conducted to investigate the effect of the key cereal grains and a microbial enzyme supplement on broiler chicken performance, gut microflora and intestinal function. Ingestion of the barley-based diet was associated with low 28-day body weight, decreased feed intake and high FCR. The supplemental enzyme increased feed intake and weight gain of the chickens on a wheat-based diet. The pH of the gizzard and caecal contents varied with the grain type. Enzyme supplementation reduced ileal viscosity, particularly in birds that received the diet based on wheat. The birds on the barley-based diet had lower ileal digestibility of dry matter, protein and energy than those given maize and sorghum-based diets. The ileal digestibility of starch was increased by enzyme supplementation. Enzyme supplementation increased the number of total anaerobic bacteria in the gizzard of birds fed on sorghum and increased lactobacilli in the gizzard of those fed both sorghum and wheat. The birds fed the sorghum-based diet had the lowest counts of caecal total anaerobic bacteria and lactobacilli. Jejunal villus height and villus:crypt ratio of birds fed the barley-based diet were the lowest when compared with those fed the other diets. Enzyme application induced an increase in villus height and villus:crypt ratio of birds on wheat, crypt depth on barley and a reduction in crypt depth of chickens on the sorghum-based diets. The highest activity of maltase and the lowest activity of sucrase were observed in tissue from birds fed on maize and sorghum-based diets respectively. The differences in the performance of broilers on cereal grains could be explained by changes in intestinal morphology, enzyme activities and gut microflora as well as nutrient digestibility. The improved performance by supplemental enzyme in wheat-fed chickens was associated with beneficial changes in intestinal morphology and digesta viscosity.

  12. MicroRNAs as regulators of drug transporters, drug-metabolizing enzymes, and tight junctions: implication for intestinal barrier function.

    PubMed

    Ikemura, Kenji; Iwamoto, Takuya; Okuda, Masahiro

    2014-08-01

    Drug transporters, drug-metabolizing enzymes, and tight junctions in the small intestine function as an absorption barrier and sometimes as a facilitator of orally administered drugs. The expression of these proteins often fluctuates and thereby causes individual pharmacokinetic variability. MicroRNAs (miRNAs), which are small non-coding RNAs, have recently emerged as a new class of gene regulator. MiRNAs post-transcriptionally regulate gene expression by binding to target mRNA to suppress its translation or regulate its degradation. They have been shown to be key regulators of proteins associated with pharmacokinetics. Moreover, the role of miRNAs on the expression of some proteins expressed in the small intestine has recently been clarified. In this review, we summarize current knowledge regarding the role of miRNAs in the regulation of drug transporters, drug-metabolizing enzymes, and tight junctions as well as its implication for intestinal barrier function. MiRNAs play vital roles in the differentiation, architecture, and barrier function of intestinal epithelial cells, and directly and/or indirectly regulate the expression and function of proteins associated with drug absorption in intestinal epithelial cells. Moreover, the variation of miRNA expression caused by pathological and physiological conditions as well as genetic factors should affect the expression of these proteins. Therefore, miRNAs could be significant factors affecting inter- and intra-individual variations in the pharmacokinetics and intestinal absorption of drugs. Overall, miRNAs could be promising targets for personalized pharmacotherapy or other attractive therapies through intestinal absorption of drugs.

  13. 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.

  14. A New Marmoset P450 4F12 Enzyme Expressed in Small Intestines and Livers Efficiently Metabolizes Antihistaminic Drug Ebastine.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Yuki, Yukako; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2016-06-01

    Common marmosets (Callithrix jacchus) are attracting attention as animal models in preclinical studies for drug development. However, cytochrome P450s (P450s), major drug-metabolizing enzymes, have not been fully identified and characterized in marmosets. In this study, based on the four novel P450 4F genes found on the marmoset genome, we successfully isolated P450 4F2, 4F3B, 4F11, and 4F12 cDNAs in marmoset livers. Deduced amino acid sequences of the four marmoset P450 4F forms exhibited high sequence identities (87%-93%) to the human and cynomolgus monkey P450 4F homologs. Marmoset P450 4F3B and 4F11 mRNAs were predominantly expressed in livers, whereas marmoset P450 4F2 and 4F12 mRNAs were highly expressed in small intestines and livers. Four marmoset P450 4F proteins heterologously expressed in Escherichia coli catalyzed the ω-hydroxylation of leukotriene B4 In addition, marmoset P450 4F12 effectively catalyzed the hydroxylation of antiallergy drug ebastine, a human P450 2J/4F probe substrate. Ebastine hydroxylation activities by small intestine and liver microsomes from marmosets and cynomolgus monkeys showed greatly higher values than those of humans. Ebastine hydroxylation activities by marmoset and cynomolgus monkey small intestine microsomes were inhibited (approximately 60%) by anti-P450 4F antibodies, unlike human small intestine microsomes, suggesting that contribution of P450 4F enzymes for ebastine hydroxylation in the small intestine might be different between marmosets/cynomolgus monkeys and humans. These results indicated that marmoset P450 4F2, 4F3B, 4F11, and 4F12 were expressed in livers and/or small intestines and were functional in the metabolism of endogenous and exogenous compounds, similar to those of cynomolgus monkeys and humans.

  15. A New Marmoset P450 4F12 Enzyme Expressed in Small Intestines and Livers Efficiently Metabolizes Antihistaminic Drug Ebastine.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Yuki, Yukako; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2016-06-01

    Common marmosets (Callithrix jacchus) are attracting attention as animal models in preclinical studies for drug development. However, cytochrome P450s (P450s), major drug-metabolizing enzymes, have not been fully identified and characterized in marmosets. In this study, based on the four novel P450 4F genes found on the marmoset genome, we successfully isolated P450 4F2, 4F3B, 4F11, and 4F12 cDNAs in marmoset livers. Deduced amino acid sequences of the four marmoset P450 4F forms exhibited high sequence identities (87%-93%) to the human and cynomolgus monkey P450 4F homologs. Marmoset P450 4F3B and 4F11 mRNAs were predominantly expressed in livers, whereas marmoset P450 4F2 and 4F12 mRNAs were highly expressed in small intestines and livers. Four marmoset P450 4F proteins heterologously expressed in Escherichia coli catalyzed the ω-hydroxylation of leukotriene B4 In addition, marmoset P450 4F12 effectively catalyzed the hydroxylation of antiallergy drug ebastine, a human P450 2J/4F probe substrate. Ebastine hydroxylation activities by small intestine and liver microsomes from marmosets and cynomolgus monkeys showed greatly higher values than those of humans. Ebastine hydroxylation activities by marmoset and cynomolgus monkey small intestine microsomes were inhibited (approximately 60%) by anti-P450 4F antibodies, unlike human small intestine microsomes, suggesting that contribution of P450 4F enzymes for ebastine hydroxylation in the small intestine might be different between marmosets/cynomolgus monkeys and humans. These results indicated that marmoset P450 4F2, 4F3B, 4F11, and 4F12 were expressed in livers and/or small intestines and were functional in the metabolism of endogenous and exogenous compounds, similar to those of cynomolgus monkeys and humans. PMID:27044800

  16. Transient changes of enzyme activities and expression of stress proteins in the small intestine of piglets after weaning.

    PubMed

    Tao, Xin; Xu, Ziwei; Men, Xiaoming

    2015-01-01

    To determine the transient effects of weaning on the small intestine, 16 piglets were slaughtered at days 0, 1, 4 and 7 after weaning. Jejunal samples were collected to examine different enzyme activities and mRNA expressions of two stress protein families, namely, heat-shock proteins (HSP) and trefoil factors (TFF). Results showed that the activities of ceruloplasmin, alkaline phosphatase and lactate dehydrogenase, were significantly changed at Day 1 and/or Day 4. The mRNA expressions of HSP10, HSP60 and HSP90 showed a pattern of increased expression with time after weaning. Expression significantly differed between Day 0 and Day 7 after weaning. The mRNA expression of HSP70 was significantly increased on Day 1 only. Similarly, the mRNA expressions of TFF1 and TFF2 were significantly increased on Day 7 compared with those on Day 0. Expression of TFF3 was not affected by time after weaning. In conclusion, the present study indicated that weaning induced transient injury to small intestinal morphology and function. Particularly it changed enzyme activities and gene expression of stress proteins in the small intestine of piglets. At first time, a change in the gene expression of HSP10 and a gene overexpression of TFF1 in the small intestine of piglets after weaning was found. PMID:25908169

  17. Transient changes of enzyme activities and expression of stress proteins in the small intestine of piglets after weaning.

    PubMed

    Tao, Xin; Xu, Ziwei; Men, Xiaoming

    2015-01-01

    To determine the transient effects of weaning on the small intestine, 16 piglets were slaughtered at days 0, 1, 4 and 7 after weaning. Jejunal samples were collected to examine different enzyme activities and mRNA expressions of two stress protein families, namely, heat-shock proteins (HSP) and trefoil factors (TFF). Results showed that the activities of ceruloplasmin, alkaline phosphatase and lactate dehydrogenase, were significantly changed at Day 1 and/or Day 4. The mRNA expressions of HSP10, HSP60 and HSP90 showed a pattern of increased expression with time after weaning. Expression significantly differed between Day 0 and Day 7 after weaning. The mRNA expression of HSP70 was significantly increased on Day 1 only. Similarly, the mRNA expressions of TFF1 and TFF2 were significantly increased on Day 7 compared with those on Day 0. Expression of TFF3 was not affected by time after weaning. In conclusion, the present study indicated that weaning induced transient injury to small intestinal morphology and function. Particularly it changed enzyme activities and gene expression of stress proteins in the small intestine of piglets. At first time, a change in the gene expression of HSP10 and a gene overexpression of TFF1 in the small intestine of piglets after weaning was found.

  18. Metabolic enzyme activities and drug excretion in the small intestine and in the liver in the rat.

    PubMed

    Almási, A; Bojcsev, Sz; Fischer, T; Simon, H; Perjési, P; Fischer, Emil

    2013-12-01

    The aim of these experiments was the investigation of the correlation between the metabolic enzyme activities and the intestinal and hepatic excretion of p-nitrophenol (PNP) and its metabolites (PNP-glucuronide: PNP-G and PNP-sulfate: PNP-S) in the same group of rats (n = 10). A jejunal loop was perfused with isotonic medium containing PNP in a concentration of 500 μM. The samples were obtained from the luminal perfusion medium and from the bile. For enzyme assays tissue samples were obtained from the liver and jejunum at the end of experiments. Significant differences were calculated by the Student's t-test. The activity of UDP-glucuronyltransferase and sulfotransferase was about three times higher in the liver than in the small intestine. The activity of the ß-glucuronidase was about six times higher, the activity of the arylsulfatase was approximately seven times greater in the liver than in the jejunum. No significant difference was found between the luminal appearance and the biliary excretion of PNP-G. Contrary to these findings, the biliary excretion of PNP-S was significantly higher than the luminal appearance of PNP-sulfate. It can be concluded that no direct correlation exists between the activity of metabolic enzymes and the excretion rate of PNP-metabolites in the liver and in the jejunal segment of the small intestine.

  19. Regulation of fatty acid elongation and initiation by acyl-acyl carrier protein in Escherichia coli.

    PubMed

    Heath, R J; Rock, C O

    1996-01-26

    Long chain acyl-acyl carrier protein (acyl-ACP) has been implicated as a physiological inhibitor of fatty acid biosynthesis since acyl-ACP degradation by thioesterase overexpression leads to constitutive, unregulated fatty acid production. The biochemical targets for acyl-ACP inhibition were unknown, and this work identified two biosynthetic enzymes that were sensitive to acyl-ACP feedback inhibition. Palmitoyl-ACP inhibited the incorporation of [14C]malonyl-CoA into long chain fatty acids in cell-free extracts of Escherichia coli. A short chain acyl-ACP species with the electrophoretic properties of beta-hydroxybutyryl-ACP accumulated concomitant with the overall decrease in the amount of [14C]malonyl-CoA incorporation, indicating that the first elongation cycle was targeted by acyl-ACP. All of the proteins required to catalyze the first round of fatty acid synthesis from acetyl-CoA plus malonyl-CoA in vitro were isolated, and the first fatty acid elongation cycle was reconstituted with these purified components. Analysis of the individual enzymes and the pattern of intermediate accumulation in the reconstituted system identified initiation of fatty acid synthesis by beta-ketoacyl-ACP synthase III (fabH) and enoyl-ACP reductase (fabI) in the elongation cycle as two steps attenuated by long chain acyl-ACP.

  20. Lipid Acyl Chain Remodeling in Yeast

    PubMed Central

    Renne, Mike F.; Bao, Xue; De Smet, Cedric H.; de Kroon, Anton I. P. M.

    2015-01-01

    Membrane lipid homeostasis is maintained by de novo synthesis, intracellular transport, remodeling, and degradation of lipid molecules. Glycerophospholipids, the most abundant structural component of eukaryotic membranes, are subject to acyl chain remodeling, which is defined as the post-synthetic process in which one or both acyl chains are exchanged. Here, we review studies addressing acyl chain remodeling of membrane glycerophospholipids in Saccharomyces cerevisiae, a model organism that has been successfully used to investigate lipid synthesis and its regulation. Experimental evidence for the occurrence of phospholipid acyl chain exchange in cardiolipin, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine is summarized, including methods and tools that have been used for detecting remodeling. Progress in the identification of the enzymes involved is reported, and putative functions of acyl chain remodeling in yeast are discussed. PMID:26819558

  1. Molecular & Biochemical Parasitology Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells

    PubMed Central

    Ringqvist, Emma; Palm, J.E. Daniel; Skarin, Hanna; Hehl, Adrian B.; Weiland, Malin; Davids, Barbara J.; Reiner, David S.; Griffiths, William J.; Eckmann, Lars; Gillin, Frances D.; Svärd, Staffan G.

    2012-01-01

    Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell–cell interactions, since only small amounts of argi-nine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine. PMID:18359106

  2. Effect of standardized cranberry extract on the activity and expression of selected biotransformation enzymes in rat liver and intestine.

    PubMed

    Bártíková, Hana; Boušová, Iva; Jedličková, Pavla; Lněničková, Kateřina; Skálová, Lenka; Szotáková, Barbora

    2014-09-18

    The use of dietary supplements containing cranberry extract is a common way to prevent urinary tract infections. As consumption of these supplements containing a mixture of concentrated anthocyanins and proanthocyanidins has increased, interest in their possible interactions with drug-metabolizing enzymes has grown. In this in vivo study, rats were treated with a standardized cranberry extract (CystiCran®) obtained from Vaccinium macrocarpon in two dosage schemes (14 days, 0.5 mg of proanthocyanidins/kg/day; 1 day, 1.5 mg of proanthocyanidins/kg/day). The aim of this study was to evaluate the effect of anthocyanins and proanthocyanidins contained in this extract on the activity and expression of intestinal and hepatic biotransformation enzymes: cytochrome P450 (CYP1A1, CYP1A2, CYP2B and CYP3A), carbonyl reductase 1 (CBR1), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Administration of cranberry extract led to moderate increases in the activities of hepatic CYP3A (by 34%), CYP1A1 (by 38%), UGT (by 40%), CBR1 (by 17%) and GST (by 13%), while activities of these enzymes in the small intestine were unchanged. No changes in the relative amounts of these proteins were found. Taken together, the interactions of cranberry extract with simultaneously administered drugs seem not to be serious.

  3. Monogalactosyldiacylglycerol biosynthesis by direct acyl transfer in Anabaena variabilis. [Anabaena variabilis

    SciTech Connect

    Chen, H.H.; Wickrema, A.; Jaworski, J.

    1987-05-01

    The authors previously reported the direct acylation of monogalactosyldiacylglycerol (MGDG) by an enzyme in the membranes of the cyanobacterium (Anabaena variabilis. The enzyme requires acyl-acyl carrier protein (acyl-ACP) as substrate, but had no other additional cofactor requirements. Palmitoyl-, stearoyl- and oleoyl-ACP were all effective substrates. The A. variabilis membranes also had a hydrolase activity which metabolized the acyl-ACP to yield free fatty acid and ACP. Possible mechanisms for the acylation reaction include either acyl exchange with existing MGDG or direct acyl transfer to a lyso-MGDG, with concomitant release of free ACP. The mechanism of this reaction has been resolved using a double labelled (/sup 14/C)acyl-(/sup 14/C)ACP substrate prepared with E. coli acyl-ACP synthetase. Following incubation with the enzyme, the unreacted (/sup 14/C)acyl-(/sup 14/C)ACP was isolated and the (/sup 14/C)acyl/(/sup 14/C)ACP ratio determined. Comparison of this ratio to that of the original substrate indicated no change and eliminated acyl exchange as a possible mechanism. Therefore, the direct acylation of lyso-MGDG is the proposed mechanism for this enzyme. The reaction is apparently specific for MGDG synthesis, as other glycolipids and phospholipids were not labelled during incubations.

  4. Histochemical distribution of digestive enzymes in the intestine of the common two-banded seabream, Diplodus vulgaris, Geoffroy St-Hilaire 1817.

    PubMed

    Tlak Gajger, I; Nejedli, S; Kozarić, Z

    2013-06-01

    The histochemical localization of non-specific esterase, alkaline and acid phosphatase as well as aminopeptidase in the intestine of the free-living common two-banded sea bream (Diplodus vulgaris) was investigated. Fish were caught near the town of Zadar (Adriatic Sea, Croatia). Samples of pyloric caeca and three parts of the intestine proper (anterior, middle and posterior) were used for the description of non-specific esterase, alkaline and acid phosphatase as well as aminopeptidase. Non-specific esterase activity was found in the cytoplasm of enterocytes in pyloric caeca and in all investigated intestinal segments. The activity was stronger in the anterior and posterior part of the intestine than in the pyloric caeca and middle segment of the intestine. Intestinal alkaline phosphatase was detected in brush border of enterocytes of all investigated intestinal segments. Enzymatic activity gradually decreased in a posterior direction. Acid phosphatase activity was observed as a fine granular reaction product in the supranuclear region of enterocytes. This activity was almost equal in pyloric caeca as well as in the anterior intestinal segment, while it was stronger in the middle and posterior intestinal segment. Aminopeptidase was present along the intestinal epithelium brush border in all investigated parts of the digestive tube. The intensity of aminopeptidase increased posteriorly. The possible role of investigated enzymes in intracellular digestion and transport is discussed.

  5. Purification of Recombinant Acyl-Coenzyme A:Cholesterol Acyltransferase 1 (ACAT1) from H293 Cells and Binding Studies Between the Enzyme and Substrates Using Difference Intrinsic Fluorescence Spectroscopy†

    PubMed Central

    Chang, Catherine CY; Miyazaki, Akira; Dong, Ruhong; Kheirollah, Alireza; Yu, Chunjiang; Geng, Yong; Higgs, Henry N; Chang, Ta-Yuan

    2010-01-01

    Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a membrane bound enzyme utilizing long-chain fatty acyl-coenzyme A and cholesterol to form cholesteryl esters and coenzyme A. Previously, we had expressed tagged human ACAT1 (hACAT1) in CHO cells and purified it to homogeneity; however, only a sparse amount of purified protein could be obtained. Here we report that the hACAT1 expression level in H293 cells is 18-fold higher than that in CHO cells. We have developed a milder purification procedure to purify the enzyme to homogeneity. The abundance of the purified protein enabled us to conduct difference intrinsic fluorescence spectroscopy to study the binding between the enzyme and its substrates in CHAPS/phospholipid mixed micelles. The results show that oleoyl CoA binds to ACAT1 with Kd=1.9 μM, and elicits significant structural changes of the protein as manifested by the significantly positive changes in its fluorescence spectrum; stearoyl CoA elicits a similar spectrum change with much lower in magnitude. Previously, kinetic studies had shown that cholesterol is an efficient substrate and an allosteric activator of ACAT1, while its diastereomer epicholesterol is neither a substrate nor an activator. Here we show that both cholesterol and epicholesterol induce positive changes in the ACAT1 fluorescence spectrum; however, the magnitude of spectrum changes induced by cholesterol is much larger than epicholesterol. These results show that stereospecificity, governed by the 3beta-OH moiety in steroid ring A, plays an important role in the binding of cholesterol to ACAT1. PMID:20964445

  6. Comparison of the accuracy and mechanism of data mining identification of the intestinal microbiota with 7 restriction enzymes.

    PubMed

    Kobayashi, Toshio; Fujiwara, Kenji

    2013-01-01

    The intestinal microbiota compositions of 92 Japanese men were identified following consumption of identical meals for 3 days, and collected feces were analyzed through terminal restriction fragment length polymorphism. The obtained operational taxonomic units (OTUs) and subjects' smoking and drinking habits, which had 2 nominal partitions, yes or no, were analyzed by Data mining software. Identification of subjects for each habit was successfully performed and reported previously, but the identification accuracy was closely dependent on the species of the applied restriction enzymes for PCR. For the sake of better selection of enzymes and understanding the mechanisms of Data mining analysis, 516f-BslI and 516f-HaeIII, 27f-MspI and 27f-AluI and 35f-HhaI, 35f-MspI and 35f-AluI, altogether 7 enzymes, were examined comparatively. Data mining analysis provides a Decision tree for identification of subjects and their dividing pathways that is produced using a limited number of OTUs, which affects the accuracy of the results. The present report discusses not only a global comparison of accuracies for characteristics, but also the detailed mechanisms that result in better or worse results and the practical roles and functions of OTUs. The OTU at the 1st step of the constructed Decision tree was the most important for any identification, and for all cases, the combination of subsequent OTUs, which formed later in the Decision tree, was also unignorable. Detailed dividing pathways were traced and compared for the 7 enzymes and the future supporting ideas were provided for better Data mining analysis of the human intestinal microbiota.

  7. Enzymatic conversion of all-trans-beta-carotene to retinal by a cytosolic enzyme from rabbit and rat intestinal mucosa.

    PubMed Central

    Lakshman, M R; Mychkovsky, I; Attlesey, M

    1989-01-01

    Enzymatic conversion of all-trans-beta-carotene to retinal by a partially purified enzyme from rabbit and rat intestinal mucosa was demonstrated. The enzymatic product was characterized based on the following evidence: (i) The product gave rise to its O-ethyloxime by treatment with O-ethylhydroxylamine with an absorption maximum at 363 nm in ethanol characteristic of authentic retinal O-ethyloxime. High-pressure liquid chromatography (HPLC) of this derivative yielded a sharp peak with a retention time of 7.99 min corresponding to the authentic compound. The enzyme blank and boiled enzyme blank failed to show any significant HPLC peaks corresponding to retinal O-ethyloxime, retinal, or retinol. (ii) The mass spectrum of the O-ethyloxime of the enzymatic product was identical to that of authentic retinal O-ethyloxime (m/z 327: 45%, M+. and m/z 282: 100%, M--ethoxy). (iii) The specific activity of the enzymatically formed [14C]retinal O-ethyloxime remained constant even after repeated crystallization. (iv) The enzymatic product exhibited an absorption maximum at 370 nm in light petroleum characteristic of authentic retinal. Furthermore, it was reduced by horse liver alcohol dehydrogenase to retinol with an absorption maximum at 326 nm in light petroleum. This retinol was enzymatically esterified to retinyl palmitate by rat pancreatic esterase with a retention time of 10 min on HPLC corresponding to authentic retinyl palmitate. Thus, the enzymatic product of beta-carotene cleavage by the partially purified intestinal enzyme was unequivocally confirmed to be retinal. PMID:2594754

  8. Enzymatic conversion of all-trans-. beta. -carotene to retinal by a cytosolic enzyme from rabbit and rat intestinal mucosa

    SciTech Connect

    Lakshman, M.R.; Mychkovsky, I.; Attlesey, M. )

    1989-12-01

    Enzymatic conversion of all-trans-{beta}-carotene to retinal by a partially purified enzyme from rabbit and rat intestinal mucosa was demonstrated. The enzymatic product was characterized based on the following evidence: (i) the product gave rise to its O-ethyloxime by treatment with O-ethylhydroxylamine with an absorption maximum at 363 nm in ethanol characteristics of authentic retinal O-ethyloxime. High-pressure liquid chromatography (HPLC) of this derivative yielded a sharp peak with a retention time of 7.99 min corresponding to the authentic compound; (ii) the mass spectrum of the O-ethyloxime of the enzymatic product was identical to that of authentic retinal O-ethyloxime; (iii) the specific activity of the enzymatically formed ({sup 14}C)retinal O-ethyloxime remained constant even after repeated crystallization; (iv) the enzymatic product exhibited an absorption maximum at 370 nm in light petroleum characteristic of authentic retinal. This retinol was enzymatically esterified to retinyl palmitate by rat pancreatic esterase with a retention time of 10 min on HPLC corresponding to authentic retinyl palmitate. Thus, the enzymatic product of {beta}-carotene cleavage by the partially purified intestinal enzyme was unequivocally confirmed to be retinal.

  9. 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

  10. Effect of orally administered betel leaf (Piper betle Linn.) on digestive enzymes of pancreas and intestinal mucosa and on bile production in rats.

    PubMed

    Prabhu, M S; Platel, K; Saraswathi, G; Srinivasan, K

    1995-10-01

    The influence of two varieties of betel leaf (Piper betle Linn.) namely, the pungent Mysore and non-pungent Ambadi, was examined on digestive enzymes of pancreas and intestinal mucosa and on bile secretion in experimental rats. The betel leaves were administered orally at two doses which were either comparable to human consumption level or 5 times this. The results indicated that while these betel leaves do not influence bile secretion and composition, they have a significant stimulatory influence on pancreatic lipase activity. Besides, the Ambadi variety of betel leaf has a positive stimulatory influence on intestinal digestive enzymes, especially lipase, amylase and disaccharidases. A slight lowering in the activity of these intestinal enzymes was seen when Mysore variety of betel leaf was administered, and this variety also had a negative effect on pancreatic amylase. Further, both the betel leaf varieties have shown decreasing influence on pancreatic trypsin and chymotrypsin activities. PMID:8575807

  11. Effect of orally administered betel leaf (Piper betle Linn.) on digestive enzymes of pancreas and intestinal mucosa and on bile production in rats.

    PubMed

    Prabhu, M S; Platel, K; Saraswathi, G; Srinivasan, K

    1995-10-01

    The influence of two varieties of betel leaf (Piper betle Linn.) namely, the pungent Mysore and non-pungent Ambadi, was examined on digestive enzymes of pancreas and intestinal mucosa and on bile secretion in experimental rats. The betel leaves were administered orally at two doses which were either comparable to human consumption level or 5 times this. The results indicated that while these betel leaves do not influence bile secretion and composition, they have a significant stimulatory influence on pancreatic lipase activity. Besides, the Ambadi variety of betel leaf has a positive stimulatory influence on intestinal digestive enzymes, especially lipase, amylase and disaccharidases. A slight lowering in the activity of these intestinal enzymes was seen when Mysore variety of betel leaf was administered, and this variety also had a negative effect on pancreatic amylase. Further, both the betel leaf varieties have shown decreasing influence on pancreatic trypsin and chymotrypsin activities.

  12. Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism.

    PubMed

    Hassaninasab, Azam; Hashimoto, Yoshiteru; Tomita-Yokotani, Kaori; Kobayashi, Michihiko

    2011-04-19

    Polyphenol curcumin, a yellow pigment, derived from the rhizomes of a plant (Curcuma longa Linn) is a natural antioxidant exhibiting a variety of pharmacological activities and therapeutic properties. It has long been used as a traditional medicine and as a preservative and coloring agent in foods. Here, curcumin-converting microorganisms were isolated from human feces, the one exhibiting the highest activity being identified as Escherichia coli. We are thus unique in discovering that E. coli was able to act on curcumin. The curcumin-converting enzyme was purified from E. coli and characterized. The native enzyme had a molecular mass of about 82 kDa and consisted of two identical subunits. The enzyme has a narrow substrate spectrum, preferentially acting on curcumin. The microbial metabolism of curcumin by the purified enzyme was found to comprise a two-step reduction, curcumin being converted NADPH-dependently into an intermediate product, dihydrocurcumin, and then the end product, tetrahydrocurcumin. We named this enzyme "NADPH-dependent curcumin/dihydrocurcumin reductase" (CurA). The gene (curA) encoding this enzyme was also identified. A homology search with the BLAST program revealed that a unique enzyme involved in curcumin metabolism belongs to the medium-chain dehydrogenase/reductase superfamily. PMID:21467222

  13. Acyl-ACP Substrate Recognition in Burkholderia mallei BmaI1 Acyl-Homoserine Lactone Synthase

    PubMed Central

    2015-01-01

    The acyl-homoserine lactone (AHL) autoinducer mediated quorum sensing regulates virulence in several pathogenic bacteria. The hallmark of an efficient quorum sensing system relies on the tight specificity in the signal generated by each bacterium. Since AHL signal specificity is derived from the acyl-chain of the acyl-ACP (ACP = acyl carrier protein) substrate, AHL synthase enzymes must recognize and react with the native acyl-ACP with high catalytic efficiency while keeping reaction rates with non-native acyl-ACPs low. The mechanism of acyl-ACP substrate recognition in these enzymes, however, remains elusive. In this study, we investigated differences in catalytic efficiencies for shorter and longer chain acyl-ACP substrates reacting with an octanoyl-homoserine lactone synthase Burkholderia mallei BmaI1. With the exception of two-carbon shorter hexanoyl-ACP, the catalytic efficiencies of butyryl-ACP, decanoyl-ACP, and octanoyl-CoA reacting with BmaI1 decreased by greater than 20-fold compared to the native octanoyl-ACP substrate. Furthermore, we also noticed kinetic cooperativity when BmaI1 reacted with non-native acyl-donor substrates. Our kinetic data suggest that non-native acyl-ACP substrates are unable to form a stable and productive BmaI1·acyl-ACP·SAM ternary complex and are thus effectively discriminated by the enzyme. These results offer insights into the molecular basis of substrate recognition for the BmaI1 enzyme. PMID:25215658

  14. The role of acyl-glucose in anthocyanin modifications.

    PubMed

    Sasaki, Nobuhiro; Nishizaki, Yuzo; Ozeki, Yoshihiro; Miyahara, Taira

    2014-11-14

    Higher plants can produce a wide variety of anthocyanin molecules through modification of the six common anthocyanin aglycons that they present. Thus, hydrophilic anthocyanin molecules can be formed and stabilized by glycosylation and acylation. Two types of glycosyltransferase (GT) and acyltransferase (AT) have been identified, namely cytoplasmic GT and AT and vacuolar GT and AT. Cytoplasmic GT and AT utilize UDP-sugar and acyl-CoA as donor molecules, respectively, whereas both vacuolar GT and AT use acyl-glucoses as donor molecules. In carnation plants, vacuolar GT uses aromatic acyl-glucoses as the glucose donor in vivo; independently, vacuolar AT uses malylglucose, an aliphatic acyl-glucose, as the acyl-donor. In delphinium and Arabidopsis, p-hydroxybenzoylglucose and sinapoylglucose are used in vivo as bi-functional donor molecules by vacuolar GT and AT, respectively. The evolution of these enzymes has allowed delphinium and Arabidopsis to utilize unique donor molecules for production of highly modified anthocyanins.

  15. The Effects of Boron Derivatives on Lipid Absorption from the Intestine and on Bile Lipids and Bile Acids of Sprague Dawley Rats

    PubMed Central

    Hall, Iris H.; Reynolds, David J.; Wong, O. T.; Sood, A.; Spielvogel, B. F.

    1995-01-01

    N,N-dimethyl-n-octadecylamine borane 1 at 8 mg/kg/day, tetrakis-u-(trimethylamine boranecarboxylato)-bis(trimethyl-carboxyborane)-dicopper(II) 2 at 2.5 mg/kg/day and trimethylamine-carboxyborane 3 at 8 mg/kg/day were evaluated for their effects on bile lipids, bile acids, small intestinal absorption of cholesterol and cholic acid and liver and small intestinal enzyme activities involved in lipid metabolism. The agent administered orally elevated rat bile excretion of lipids, e.g. cholesterol and phospholipids, and compounds 2 and 3 increased the bile flow rate. These agents altered the composition of the bile acids, but there was no significant increase in lithocholic acid which is most lithogenic agent in rats. The three agents did decrease cholesterol absorption from isolated in situ intestinal duodenum loops in the presence of drug. Hepatic and small intestinal mucosa enzyme activities, e.g. ATP-dependent citrate lyase, acyl CoA cholesterol acyl transferase, cholsterol-7-α -hydroxylase, sn glycerol-3-phosphate acyl transferase, phosphatidylate phosphohydrolase, and lipoprotein lipase, were reduced. However, the boron derivatives 1 and 3 decreased hepatic HMG-CoA reductase activity, the regulatory enzyme for cholesterol synthesis, but the compounds had no effects on small intestinal mucosa HMG-CoA reductase activity. There was no evidence of hepatic cell damage afforded by the drugs based on clinical chemistry values which would induce alterations in bile acid concentrations after treatment of the rat. PMID:18472747

  16. Effect of dietary supplementation with an ethanolic extract of propolis on broiler intestinal morphology and digestive enzyme activity.

    PubMed

    Eyng, C; Murakami, A E; Duarte, C R A; Santos, T C

    2014-04-01

    The present study aimed to evaluate the effect of different levels of an ethanolic extract of propolis (EEP) on broiler performance, carcass characteristics, weight of gastrointestinal organs, intestinal morphometry and digestive enzyme activity. 1020 male broiler chicks were assigned in a completely randomised experimental design to six treatments (EEP supplement levels of 0, 1000, 2000, 3000, 4000 and 5000 ppm) and five replications, and 34 birds per experimental unit. The experimental diets were administered from 1 to 21 days of age, and the birds were subsequently provided a ration based on corn and soybean meal. EEP supplementation from 1 to 7 days negatively affected (p < 0.05) the weight gain and feed intake. The proventriculus weight at 7 days exhibited a quadratic response (p < 0.05), which predicted a lower weight at a dose of 2865 ppm of the EEP. For the duodenum at 21 days of age, the response pattern (p < 0.05) predicted that birds that were fed 2943 and 3047 ppm of the EEP would exhibit an improved crypt depth and villus-to-crypt ratio respectively. The villus height, crypt depth and villus-to-crypt ratio in the jejunum and the ileum were not affected (p > 0.05). With increased EEP doses, the duodenal sucrase activity linearly decreased at 7 days of age and linearly increased in the jejunum at 21 days of age (p < 0.05), while pancreatic enzyme activity was unaffected (p > 0.05). Although the carcass and cut yields did not improve, the percentage of abdominal fat decreased (p < 0.05). The supplementation of the broiler pre-starter diet with 1000-5000 ppm of the EEP impaired performance at this stage, most likely due to the decreased sucrase activity. However, the EEP supplementation from 3000 ppm improved intestinal morphophysiology at 21 days of age and did not affect the performance or carcass yield at 42 days of age.

  17. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  18. Vertebrate Acyl CoA synthetase family member 4 (ACSF4-U26) is a β-alanine-activating enzyme homologous to bacterial non-ribosomal peptide synthetase.

    PubMed

    Drozak, Jakub; Veiga-da-Cunha, Maria; Kadziolka, Beata; Van Schaftingen, Emile

    2014-03-01

    Mammalian ACSF4-U26 (Acyl CoA synthetase family member 4), a protein of unknown function, comprises a putative adenylation domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site, and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 show greater identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was not formed in a point mutant lacking the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was almost specific for β-alanine, and a KM of ~ 5 μm was calculated for this reaction. The loaded enzyme was used to study the formation of a potential end product. Among the 20 standard amino acids, only cysteine stimulated unloading of the enzyme. This effect was mimicked by cysteamine and dithiothreitol, and was unaffected by absence of the PQQDH-related domain, suggesting that β-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification.

  19. 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.

  20. Technical Aspects of Nominal Partitions on Accuracy of Data Mining Classification of Intestinal Microbiota - Comparison between 7 Restriction Enzymes.

    PubMed

    Kobayashi, Toshio; Fujiwara, Kenji

    2014-01-01

    The application of data mining analyses (DM) is effective for the quantitative classification of human intestinal microbiota (HIM). However, there remain various technical problems that must be overcome. This paper deals with the number of nominal partitions (NP) of the target dataset, which is a major technical problem. We used here terminal restriction fragment length polymorphism data, which was obtained from the feces of 92 Japanese men. Data comprised operational taxonomic units (OTUs) and subject smoking and drinking habits, which were effectively classified by two NP (2-NP; Yes or No). Using the same OTU data, 3-NP and 5-NP were examined here and results were obtained, focusing on the accuracies of prediction, and the reliability of the selected OTUs by DM were compared to the former 2-NP. Restriction enzymes for PCR were further affected by the accuracy and were compared with 7 enzymes. There were subjects who possess HIM at the border zones of partitions, and the greater the number of partitions, the lower the obtained DM accuracy. The application of balance nodes boosted and duplicated the data, and was able to improve accuracy. More accurate and reliable DM operations are applicable to the classification of unknown subjects for identifying various characteristics, including disease.

  1. Digestive enzyme activity in the intestine of Nile tilapia (Oreochromis niloticus L.) under pond and cage farming systems.

    PubMed

    Santos, Juliana Ferreira; Soares, Karollina Lopes Siqueira; Assis, Caio Rodrigo Dias; Guerra, Carlos Augusto Martins; Lemos, Daniel; Carvalho, Luiz Bezerra; Bezerra, Ranilson Souza

    2016-10-01

    The effect of different farming systems (cage, pond) upon digestive enzyme activities of Nile tilapia was evaluated. Juvenile Nile tilapia (87.61 ± 1.52 g) were simultaneously cultured in pond and cage systems during 90 days. Cages used nutritional biphasic plan (35 and 32 % crude protein-CP feeds) and ponds used nutritional triphasic plan (35, 32 and 28 % CP feeds). Biometric measurements were monthly performed for adjustments in feeding regimes and removal of intestine tissues to evaluate the performance of enzyme activities. Total proteolytic, amylase and lipase activities were not statistically different between the treatments throughout the periods analyzed (31, 63 and 94 days of culture). However, trypsin and chymotrypsin activities were higher with 31 and 63 days of culture in fish from pond system, suggesting that natural food may have influenced these activities. A positive correlation was observed between the recommended concentration of essential amino acids for Nile tilapia and specific aminopeptidases activity in fish cage system. Substrate-SDS-PAGE revealed 12 active proteolytic bands in both systems. However, integrated density (ID) values were higher in the bands of ponds. Specimens of either cage or pond exhibited five bands of amylolytic activity. Fish from cage and pond systems showed the highest values of ID within 31 days of cultivation. In this study, the complexity of digestive functions could be verified for animals maintained under commercial conditions. Some of the assessed enzymes may show adaptations of their activities and/or expression that allow the fish to achieve a more efficient nutrient assimilation. PMID:27021899

  2. Versatility of acyl-acyl carrier protein synthetases.

    PubMed

    Beld, Joris; Finzel, Kara; Burkart, Michael D

    2014-10-23

    The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. We show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E. coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. In vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms. PMID:25308274

  3. Evaluation of the synergistic effect of Allium sativum, Eugenia jambolana, Momordica charantia, Ocimum sanctum, and Psidium guajava on hepatic and intestinal drug metabolizing enzymes in rats

    PubMed Central

    Kumar, Devendra; Trivedi, Neerja; Dixit, Rakesh K.

    2016-01-01

    Aims/Background: This study was to investigated the synergistic effect of polyherbal formulations (PHF) of Allium sativum L., Eugenia jambolana Lam., Momordica charantia L., Ocimum sanctum Linn., and Psidium guajava L. in the inhibition/induction of hepatic and intestinal cytochrome P450 (CYPs) and Phase-II conjugated drug metabolizing enzymes (DMEs). Consumption of these herbal remedy has been extensively documented for diabetes treatment in Ayurveda. Methodology: PHF of these five herbs was prepared, and different doses were orally administered to Sprague–Dawley rats of different groups except control group. Expression of mRNA and activity of DMEs were examined by real-time polymerase chain reaction and high performance liquid chromatography in isolated liver and intestine microsomes in PHF pretreated rats. Results: The activities of hepatic and intestinal Phase-II enzyme levels increased along with mRNA levels except CYP3A mRNA level. PHF administration increases the activity of hepatic and intestinal UDP-glucuronyltransferase and glutathione S-transferase in response to dose and time; however, the activity of hepatic sulfotransferase increased at higher doses. Conclusions: CYPs and Phase-II conjugated enzymes levels can be modulated in dose and time dependent manner. Observations suggest that polyherbal formulation might be a possible cause of herb-drug interaction, due to changes in pharmacokinetic of crucial CYPs and Phase-II substrate drug. PMID:27757267

  4. Comparison of pH-dependent allostery and dissociation for phosphofructokinases from Artemia embryos and rabbit muscle: nature of the enzymes acylated with diethylpyrocarbonate.

    PubMed

    Carpenter, J F; Hand, S C

    1986-07-01

    Purified Artemia phosphofructokinase (PFK), unlike the rabbit skeletal muscle enzyme, displays allosteric kinetics at pH 8, a feature that is functionally significant since the intracellular pH of the developing brine shrimp embryo is greater than or equal to 7.9. Catalytic activity of the Artemia enzyme is severely suppressed by acidic pH even when assayed at the adenylate nucleotide concentrations existing in anaerobic embryos, which is consistent with the lack of a Pasteur effect in these organisms. For both PFK homologs, carbethoxylation reduces the sensitivity to ATP and citrate inhibition, the cooperativity as a function of fructose 6-phosphate concentration and the degree of activation in the presence ADP, AMP, and fructose 2,6-bisphosphate. Considering the role of histidine protonation in PFK allosteric control, the capacity for regulatory kinetics seen at pH 8 in the Artemia enzyme could be explained in part by upward shifts in pKa values of ionizable residues. pH-induced dissociation of tetrameric Artemia PFK into inactive subunits does not occur during catalytic inhibition at acidic pH (pH 6.5, 6 degrees C), as judged by 90 degree light scattering. This observation contrasts markedly with the dimerization and inactivation of rabbit PFK, but is shown not to be unique when compared to other selected PFK homologs. Neither the acute pH sensitivity of Artemia PFK nor the pH-induced hysteretic inactivation displayed by the rabbit enzyme are altered by carbethoxylation, suggesting that ionizable residues involved in these two processes are not the same ones involved in allosteric kinetics. PMID:2942107

  5. Identification and functional expression of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals.

    PubMed

    Kroon, Johan T M; Wei, Wenxue; Simon, William J; Slabas, Antoni R

    2006-12-01

    Seed oil from castor bean (Ricinus communis) contains high amounts of hydroxy fatty acid rich triacylglycerols (TAGs) that can serve as raw material for production of bio-based products such as nylon, cosmetics, lubricants, foams, and surfactants. Diacylglycerol acyltransferase (DGAT) catalyses the terminal reaction in the acyl-CoA dependent Kennedy pathway of triglyceride biosynthesis. There is still some debate whether there are three or four enzymes in yeast that have DGAT activity and catalyse the synthesis of TAG but of these the DGAT2 homologue Dga1 contributes in a major way to TAG biosynthesis. Here we report on the cloning of a cDNA for DGAT2 from castor bean and prove its biological activity following expression in yeast and enzymatic assays using diricinolein as the acceptor and ricinoleoyl-CoA as the donor. Previous reports of DGAT in castor have focussed on DGAT1 which has little amino acid sequence homology to DGAT2. Expressional studies demonstrate that DGAT2 is 18-fold more highly expressed in seeds than in leaves and shows temporal specific expression during seed development. In contrast, DGAT1 shows little difference in expression in seeds versus leaves. We conclude that in castor bean DGAT2 is more likely to play a major role in seed TAG biosynthesis than DGAT1.

  6. High-Moisture Diet for Laboratory Rats: Complete Blood Counts, Serum Biochemical Values, and Intestinal Enzyme Activity

    NASA Technical Reports Server (NTRS)

    Battles, August H.; Knapka, Joseph T.; Stevens, Bruce R.; Lewis, Laura; Lang, Marie T.; Gruendel, Douglas J.

    1991-01-01

    Rats were fed an irradiated high-moisture diet (KSC-25) with or without access to a water bottle. Physiologic values were compared between these two groups and a group of rats fed a purified diet. Hematologic and serum biochemical values, urine specific gravity, and intestinal enzyme activities were determined from samples collected from the three groups of rats. Sprague Dawley rats (n=32) fed the irradiated high-moisture diet with or without a water bottle were the test animals. Rats (n=16) fed an irradiated purified diet and water provided via a water bottle were the control group. The purified diet formulation, modified AIN-76A, is a commonly used purified diet for laboratory rodents. All rats remained alert and healthy throughout the study. A comparison of the physiologic values of rats in this study with reported normal values indicated that all of the rats in the study were in good health. Significant differences (P less than 0.05) of the physiologic values from each rat group are reported.

  7. Stereoselective metabolism of carvedilol by the beta-naphthoflavone-inducible enzyme in human intestinal epithelial Caco-2 cells.

    PubMed

    Ishida, Kazuya; Honda, Mutsuko; Shimizu, Takako; Taguchi, Masato; Hashimoto, Yukiya

    2007-10-01

    Treatment of Caco-2 cells with beta-naphthoflavone (beta-NF) and 1alpha,25-dihydroxyvitamin D(3) (VD(3)) induces UDP-glucuronosyltransferases (UGTs) and cytochrome P450 (CYP) 3A4, respectively. In the present study, we evaluated the metabolism of carvedilol in beta-NF- and VD(3)-treated Caco-2 cells. The metabolism of R-carvedilol was not significant in non-treated Caco-2 cells, whereas S-carvedilol was significantly metabolized in the cells. The metabolism of R- and S-carvedilol was significantly increased by the treatment of Caco-2 cells with 50 microM beta-NF for 3 d. In contrast, the treatment of Caco-2 cells with 250 nM VD(3) for 2 weeks did not induce a significant change in the metabolism of R- and S-carvedilol. The metabolism of carvedilol in beta-NF-treated Caco-2 cells was markedly inhibited by a substrate of UGTs, baicalein. In addition, the expression of UGT1A1, 1A6, and 1A9 mRNA was increased in beta-NF-treated Caco-2 cells as compared with non-treated cells. These findings indicated that carvedilol was metabolized stereoselectively by the beta-NF-inducible enzyme in Caco-2 cells. The UGT1A subfamily in intestinal epithelial cells may be partly responsible for first-pass (presystemic) metabolism of the drug. PMID:17917264

  8. Lysine fatty acylation promotes lysosomal targeting of TNF-α

    PubMed Central

    Jiang, Hong; Zhang, Xiaoyu; Lin, Hening

    2016-01-01

    Tumor necrosis factor-α (TNF-α) is a proinflammation cytokine secreted by various cells. Understanding its secretive pathway is important to understand the biological functions of TNF-α and diseases associated with TNF-α. TNF-α is one of the first proteins known be modified by lysine fatty acylation (e.g. myristoylation). We previously demonstrated that SIRT6, a member of the mammalian sirtuin family of enzymes, can remove the fatty acyl modification on TNF-α and promote its secretion. However, the mechanistic details about how lysine fatty acylation regulates TNF-α secretion have been unknown. Here we present experimental data supporting that lysine fatty acylation promotes lysosomal targeting of TNF-α. The result is an important first step toward understanding the biological functions of lysine fatty acylation. PMID:27079798

  9. Effect of dietary arginine on growth, intestinal enzyme activities and gene expression in muscle, hepatopancreas and intestine of juvenile Jian carp (Cyprinus carpio var. Jian).

    PubMed

    Chen, Gangfu; Feng, Lin; Kuang, Shengyao; Liu, Yang; Jiang, Jun; Hu, Kai; Jiang, Weidan; Li, Shuhong; Tang, Ling; Zhou, Xiaoqiu

    2012-07-01

    The present study was conducted to test the hypothesis that dietary arginine promotes digestion and absorption capacity, and, thus, enhances fish growth. This improvement might be related to the target of rapamycin (TOR) and eIF4E-binding protein (4E-BP). A total of 1200 juvenile Jian carp, Cyprinus carpio var. Jian, with an average initial weight of 6.33 (SE 0.03) g, were fed with diets containing graded concentrations of arginine, namely, 9.8 (control), 12.7, 16.1, 18.5, 21.9 and 24.5 g arginine/kg diet for 9 weeks. An real-time quantitative PCR analysis was performed to determine the relative expression of TOR and 4E-BP in fish muscle, hepatopancreas and intestine. Dietary arginine increased (P < 0.05): (1) glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase activities in muscle and hepatopancreas; (2) intestine and hepatopancreas protein content, folds height, and trypsin, chymotrypsin, lipase, Na⁺/K⁺-ATPase, alkaline phosphatase, γ-glutamyl transpeptidase and creatine kinase activities in intestine; (3) Lactobacillus counts; (4) relative expression of TOR in the muscle, hepatopancreas and distal intestine (DI); (5) relative expression of 4E-BP in proximal intestine (PI) and mid-intestine (MI), as compared with the control group. In contrast, dietary arginine reduced (P < 0.05): (1) plasma ammonia content; (2) Aeromonas hydrophila and Escherichia coli counts; (3) relative expression of TOR in PI and MI; (4) relative expression of 4E-BP in the muscle, hepatopancreas and DI. The arginine requirement estimated by specific growth rate using quadratic regression analysis was found to be 18.0 g/kg diet. These results indicate that arginine improved fish growth, digestive and absorptive ability and regulated the expression of TOR and 4E-BP genes.

  10. Extrusion decreases the negative effects of kidney bean on enzyme and transport activities of the rat small intestine.

    PubMed

    Marzo, F; Milagro, F I; Urdaneta, E; Barrenetxe, J; Ibañez, F C

    2011-10-01

    The objective of the present study was to evaluate the influence of raw and extruded kidney bean (Phaseolus vulgaris L. var. Pinto) consumption on the gut physiology of young growing rats. The intestinal enzyme activity (sucrase, maltase, Na(+) /K(+) ATPase, aminopeptidase N, dipeptidylpeptidase IV, alkaline phosphatase) and the uptake of sugar (d-galactose) and amino acids (l-leucine) were measured in brush border membrane vesicles. Five groups of growing male Wistar rats were fed ad libitum for 15 days on five different 10% protein diets: one containing casein as the main source of protein (Control, C), and four containing raw (RKB1, RKB6) or extruded kidney bean (EKB1, EKB6) at 1% and 6% of total protein content respectively. Extrusion treatment significantly reduced the content of bioactive factors (phytates, tannins) and abolished lectins, trypsin, chymotrypsin, and α-amylase inhibitory activities. Rats fed raw beans (especially RKB6) showed lower growth rate and food intake as compared to those fed extruded legumes, probably due to the high levels of lectins and other anti-nutritive factors in the raw beans. Gut enzymatic activities and uptake of d-galactose and l-leucine were lower in RKB6 and RKB1-fed animals, although they significantly improved in the groups fed extruded beans. Enzymatic activity and uptake in EKB1 were similar to those of casein-fed rats, whereas the uptake and growth rate of EKB6 were different to the control. This is attributable to the higher non-thermolabile biofactor content in the EKB6 diet, especially phytates and tannins, than in EKB1. This article shows the dose-dependent toxicological effects of bioactive factors contained in kidney beans on gut function. The extrusion process reduced their adverse impact on gut physiology and growth rate.

  11. Extrusion decreases the negative effects of kidney bean on enzyme and transport activities of the rat small intestine.

    PubMed

    Marzo, F; Milagro, F I; Urdaneta, E; Barrenetxe, J; Ibañez, F C

    2011-10-01

    The objective of the present study was to evaluate the influence of raw and extruded kidney bean (Phaseolus vulgaris L. var. Pinto) consumption on the gut physiology of young growing rats. The intestinal enzyme activity (sucrase, maltase, Na(+) /K(+) ATPase, aminopeptidase N, dipeptidylpeptidase IV, alkaline phosphatase) and the uptake of sugar (d-galactose) and amino acids (l-leucine) were measured in brush border membrane vesicles. Five groups of growing male Wistar rats were fed ad libitum for 15 days on five different 10% protein diets: one containing casein as the main source of protein (Control, C), and four containing raw (RKB1, RKB6) or extruded kidney bean (EKB1, EKB6) at 1% and 6% of total protein content respectively. Extrusion treatment significantly reduced the content of bioactive factors (phytates, tannins) and abolished lectins, trypsin, chymotrypsin, and α-amylase inhibitory activities. Rats fed raw beans (especially RKB6) showed lower growth rate and food intake as compared to those fed extruded legumes, probably due to the high levels of lectins and other anti-nutritive factors in the raw beans. Gut enzymatic activities and uptake of d-galactose and l-leucine were lower in RKB6 and RKB1-fed animals, although they significantly improved in the groups fed extruded beans. Enzymatic activity and uptake in EKB1 were similar to those of casein-fed rats, whereas the uptake and growth rate of EKB6 were different to the control. This is attributable to the higher non-thermolabile biofactor content in the EKB6 diet, especially phytates and tannins, than in EKB1. This article shows the dose-dependent toxicological effects of bioactive factors contained in kidney beans on gut function. The extrusion process reduced their adverse impact on gut physiology and growth rate. PMID:21114542

  12. Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20 Expression

    PubMed Central

    Murata, Kozue; Chiba, Eriko; Shimazu, Tomoyuki; Aso, Hisashi; Iwabuchi, Noriyuki; Xiao, Jin-zhong; Saito, Tadao; Kitazawa, Haruki

    2013-01-01

    Background We previously showed that evaluation of anti-inflammatory activities of lactic acid bacteria in porcine intestinal epithelial (PIE) cells is useful for selecting potentially immunobiotic strains. Objective The aims of the present study were: i) to select potentially immunomodulatory bifidobacteria that beneficially modulate the Toll-like receptor (TLR)-4-triggered inflammatory response in PIE cells and; ii) to gain insight into the molecular mechanisms involved in the anti-inflammatory effect of immunobiotics by evaluating the role of TLR2 and TLR negative regulators in the modulation of proinflammatory cytokine production and activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways in PIE cells. Results Bifidobacteria longum BB536 and B. breve M-16V strains significantly downregulated levels of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and IL-6 in PIE cells challenged with heat-killed enterotoxigenic Escherichia coli. Moreover, BB536 and M-16V strains attenuated the proinflammatory response by modulating the NF-κB and MAPK pathways. In addition, our findings provide evidence for a key role for the ubiquitin-editing enzyme A20 in the anti-inflammatory effect of immunobiotic bifidobacteria in PIE cells. Conclusions We show new data regarding the mechanism involved in the anti-inflammatory effect of immunobiotics. Several strains with immunoregulatory capabilities used a common mechanism to induce tolerance in PIE cells. Immunoregulatory strains interacted with TLR2, upregulated the expression of A20 in PIE cells, and beneficially modulated the subsequent TLR4 activation by reducing the activation of MAPK and NF-κB pathways and the production of proinflammatory cytokines. We also show that the combination of TLR2 activation and A20 induction can be used as biomarkers to screen and select potential immunoregulatory bifidobacteria strains. PMID:23555642

  13. Mapping the intestinal alpha-glucogenic enzyme specificities of starch digesting maltase-glucoamylase and sucrase-isomaltase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of intestinal alpha-glucosidases and pancreatic alpha-amylases is an approach to controlling blood glucose and serum insulin levels in individuals with Type II diabetes. The two human intestinal glucosidases are maltase-glucoamylase and sucrase-isomaltase. Each incorporates two family 31 ...

  14. Development of an antigen-capture enzyme-linked immunosorbent assay for Clostridium perfringens beta2-toxin in porcine feces and the neonatal piglet intestine.

    PubMed

    Kircanski, Jasmina; Hodgins, Douglas; Soltes, Glenn; Pei, Yanlong; Parreira, Valeria R; Songer, J Glenn; Prescott, John F

    2012-09-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for detection and quantitation of beta2-toxin in neonatal piglet intestinal contents. Polystyrene plates were coated with polyclonal capture antibodies prepared against consensus recombinant beta2-toxin. The ELISA was developed using consensus recombinant beta2-toxin, atypical recombinant beta2-toxin, purified consensus native beta2-toxin, and field samples of neonatal porcine intestinal contents. Captured antigen was detected using a horseradish peroxidase-labeled monoclonal antibody against consensus recombinant beta2-toxin. The limit of detection of the ELISA for consensus beta2-toxin was between 2.0 and 3.5 ng/ml. The ELISA detected atypical recombinant beta2-toxin only weakly. Optical density was protein concentration dependent. The test confirmed differences between consensus and atypical recombinant beta2-toxin, but similar results obtained when testing pure consensus recombinant beta2-toxin and native beta2-toxin. Results obtained from intestinal content samples, particularly from the small intestine, were highly inconsistent and suggested variable protease activity. Addition of protease inhibitors partially prevented degradation of the toxin; however, sample processing at low temperature, at a lower pH (citrate buffer with 5% of bovine serum albumin, pH 6.1), and "cold incubation" of applied antigens abolished protease activity. The recombinant toxin was preserved in spiked intestinal samples by freezing at -70°C, suggesting that necropsy samples can be stored frozen for periodic testing. With appropriate sample preparation, antigen-capture ELISA can detect beta2-toxin in the intestinal content and feces of neonatal piglets.

  15. GOAT induced ghrelin acylation regulates hedonic feeding.

    PubMed

    Davis, J F; Perello, M; Choi, D L; Magrisso, I J; Kirchner, H; Pfluger, P T; Tschoep, M; Zigman, J M; Benoit, S C

    2012-11-01

    Ghrelin is an orexigenic hormone that regulates homeostatic and reward-related feeding behavior. Recent evidence indicates that acylation of ghrelin by the gut enzyme ghrelin O-acyl transferase (GOAT) is necessary to render ghrelin maximally active within its target tissues. Here we tested the hypothesis that GOAT activity modulates food motivation and food hedonics using behavioral pharmacology and mutant mice deficient for GOAT and the ghrelin receptor (GHSR). We evaluated operant responding following pharmacological administration of acyl-ghrelin and assessed the necessity of endogenous GOAT activity for operant responding in GOAT and GHSR-null mice. Hedonic-based feeding behavior also was examined in GOAT-KO and GHSR-null mice using a "Dessert Effect" protocol in which the intake of a palatable high fat diet "dessert" was assessed in calorically-sated mice. Pharmacological administration of acyl-ghrelin augmented operant responding; notably, this effect was dependent on intact GHSR signaling. GOAT-KO mice displayed attenuated operant responding and decreased hedonic feeding relative to controls. These behavioral results correlated with decreased expression of the orexin-1 receptor in reward-related brain regions in GOAT-KO mice. In summary, the ability of ghrelin to stimulate food motivation is dependent on intact GHSR signaling and modified by endogenous GOAT activity. Furthermore, GOAT activity is required for hedonic feeding behavior, an effect potentially mediated by forebrain orexin signaling. These data highlight the significance of the GOAT-ghrelin system for the mediation of food motivation and hedonic feeding.

  16. Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence

    SciTech Connect

    Byers, D.M.; Meighen, E.A.

    1985-09-01

    Pulse-chase experiments with (/sup 3/H)tetradecanoic acid and ATP showed that the bioluminescence-related 32-kDa acyltransferase from Vibrio harveyi can specifically catalyze the deacylation of a /sup 3/H-labeled 18-kDa protein observed in extracts of this bacterium. The 18-kDa protein has been partially purified and its physical and chemical properties strongly indicate that it is fatty acyl-acyl carrier protein (acyl-ACP). Both this V. harveyi (/sup 3/H)acylprotein and (/sup 3/H)palmitoyl-ACP from Escherichia coli were substrates in vitro for either the V. harveyi 32-kDa acyltransferase or the analogous enzyme (34K) from Photobacterium phosphoreum. TLC analysis indicated that the hexane-soluble product of the reaction is fatty acid. No significant cleavage of either E. coli or V. harveyi tetradecanoyl-ACP was observed in extracts of these bacteria unless the 32-kDa or 34K acyltransferase was present. Since these enzymes are believed to be responsible for the supply of fatty acids for reduction to form the aldehyde substrate of luciferase, the above results suggest that long-chain acyl-ACP is the source of fatty acids for bioluminescence.

  17. 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

  18. Intestinal steroidogenesis.

    PubMed

    Bouguen, Guillaume; Dubuquoy, Laurent; Desreumaux, Pierre; Brunner, Thomas; Bertin, Benjamin

    2015-11-01

    Steroids are fundamental hormones that control a wide variety of physiological processes such as metabolism, immune functions, and sexual characteristics. Historically, steroid synthesis was considered a function restricted to the adrenals and the gonads. In the past 20 years, a significant number of studies have demonstrated that steroids could also be synthesized or metabolized by other organs. According to these studies, the intestine appears to be a major source of de novo produced glucocorticoids as well as a tissue capable of producing and metabolizing sex steroids. This finding is based on the detection of steroidogenic enzyme expression as well as the presence of bioactive steroids in both the rodent and human gut. Within the intestinal mucosa, the intestinal epithelial cell layer is one of the main cellular sources of steroids. Glucocorticoid synthesis regulation in the intestinal epithelial cells is unique in that it does not involve the classical positive regulator steroidogenic factor-1 (SF-1) but a closely related homolog, namely the liver receptor homolog-1 (LRH-1). This local production of immunoregulatory glucocorticoids contributes to intestinal homeostasis and has been linked to pathophysiology of inflammatory bowel diseases. Intestinal epithelial cells also possess the ability to metabolize sex steroids, notably estrogen; this mechanism may impact colorectal cancer development. In this review, we contextualize and discuss what is known about intestinal steroidogenesis and regulation as well as the key role these functions play both in physiological and pathological conditions.

  19. Short-term oral exposure to aluminium decreases glutathione intestinal levels and changes enzyme activities involved in its metabolism.

    PubMed

    Orihuela, Daniel; Meichtry, Verónica; Pregi, Nicolás; Pizarro, Manuel

    2005-09-01

    To study the effects of aluminium (Al) on glutathione (GSH) metabolism in the small intestine, adult male Wistar rats were orally treated with AlCl3.6H2O at doses of 30, 60, 120 and 200 mg/kg body weight (b.w.) per day, during seven days. Controls received deionized water. At doses above 120 mg/kg b.w., Al produced both a significant reduction of GSH content and an increase of oxidized/reduced glutathione ratio (P < 0.05). The index of oxidative stress of the intestine mucosa in terms of lipid peroxidation evaluated by thiobarbituric acid reactive substances was significantly increased (52%) at higher Al dose used. The duodenal expression of the multidrug resistance-associated protein 2 in brush border membranes, determined by Western blot technique, was increased 2.7-fold in rats treated with 200mg AlCl3/kg b.w (P < 0.01). Intestine activities of both GSH-synthase (from 60 mg/kg b.w.) and GSSG-reductase (from 120 mg/kg b.w.) were significantly reduced (26% and 31%, respectively) while glutathione-S-transferase showed to be slightly modified in the Al-treated groups. Conversely, gamma-glutamyltranspeptidase activity was significantly increased (P < 0.05) due to the Al treatment. Al reduced in vitro mucosa-to-lumen GSH efflux (P < 0.05). A positive linear correlation between the intestine GSH depletion and reduction of in situ 45Ca intestinal absorption, both produced by Al, was found (r = 0.923, P = 0.038). Taking as a whole, these results show that Al would alter GSH metabolism in small intestine by decreasing its turnover, leading to an unbalance of redox state in the epithelial cells, thus contributing to deteriorate GSH-dependent absorptive functions. PMID:16084594

  20. Short-term oral exposure to aluminium decreases glutathione intestinal levels and changes enzyme activities involved in its metabolism.

    PubMed

    Orihuela, Daniel; Meichtry, Verónica; Pregi, Nicolás; Pizarro, Manuel

    2005-09-01

    To study the effects of aluminium (Al) on glutathione (GSH) metabolism in the small intestine, adult male Wistar rats were orally treated with AlCl3.6H2O at doses of 30, 60, 120 and 200 mg/kg body weight (b.w.) per day, during seven days. Controls received deionized water. At doses above 120 mg/kg b.w., Al produced both a significant reduction of GSH content and an increase of oxidized/reduced glutathione ratio (P < 0.05). The index of oxidative stress of the intestine mucosa in terms of lipid peroxidation evaluated by thiobarbituric acid reactive substances was significantly increased (52%) at higher Al dose used. The duodenal expression of the multidrug resistance-associated protein 2 in brush border membranes, determined by Western blot technique, was increased 2.7-fold in rats treated with 200mg AlCl3/kg b.w (P < 0.01). Intestine activities of both GSH-synthase (from 60 mg/kg b.w.) and GSSG-reductase (from 120 mg/kg b.w.) were significantly reduced (26% and 31%, respectively) while glutathione-S-transferase showed to be slightly modified in the Al-treated groups. Conversely, gamma-glutamyltranspeptidase activity was significantly increased (P < 0.05) due to the Al treatment. Al reduced in vitro mucosa-to-lumen GSH efflux (P < 0.05). A positive linear correlation between the intestine GSH depletion and reduction of in situ 45Ca intestinal absorption, both produced by Al, was found (r = 0.923, P = 0.038). Taking as a whole, these results show that Al would alter GSH metabolism in small intestine by decreasing its turnover, leading to an unbalance of redox state in the epithelial cells, thus contributing to deteriorate GSH-dependent absorptive functions.

  1. Possible Role of Different Yeast and Plant Lysophospholipid:Acyl-CoA Acyltransferases (LPLATs) in Acyl Remodelling of Phospholipids.

    PubMed

    Jasieniecka-Gazarkiewicz, Katarzyna; Demski, Kamil; Lager, Ida; Stymne, Sten; Banaś, Antoni

    2016-01-01

    Recent results have suggested that plant lysophosphatidylcholine:acyl-coenzyme A acyltransferases (LPCATs) can operate in reverse in vivo and thereby catalyse an acyl exchange between the acyl-coenzyme A (CoA) pool and the phosphatidylcholine. We have investigated the abilities of Arabidopsis AtLPCAT2, Arabidopsis lysophosphatidylethanolamine acyltransferase (LPEAT2), S. cerevisiae lysophospholipid acyltransferase (Ale1) and S. cerevisiae lysophosphatidic acid acyltransferase (SLC1) to acylate lysoPtdCho, lysoPtdEtn and lysoPtdOH and act reversibly on the products of the acylation; the PtdCho, PtdEtn and PtdOH. The tested LPLATs were expressed in an S. cervisiae ale1 strain and enzyme activities were assessed in assays using microsomal preparations of the different transformants. The results show that, despite high activity towards lysoPtdCho, lysoPtdEtn and lysoPtdOH by the ALE1, its capacities to operate reversibly on the products of the acylation were very low. Slc1 readily acylated lysoPtdOH, lysoPtdCho and lysoPtdEtn but showed no reversibility towards PtdCho, very little reversibility towards PtdEtn and very high reversibility towards PtdOH. LPEAT2 showed the highest levels of reversibility towards PtdCho and PtdEtn of all LPLATs tested but low ability to operate reversibly on PtdOH. AtLPCAT2 showed good reversible activity towards PtdCho and PtdEtn and very low reversibility towards PtdOH. Thus, it appears that some of the LPLATs have developed properties that, to a much higher degree than other LPLATs, promote the reverse reaction during the same assay conditions and with the same phospholipid. The results also show that the capacity of reversibility can be specific for a particular phospholipid, albeit the lysophospholipid derivatives of other phospholipids serve as good acyl acceptors for the forward reaction of the enzyme. PMID:26643989

  2. Effect of apelin on mitosis, apoptosis and DNA repair enzyme OGG 1/2 expression in intestinal cell lines IEC-6 and Caco-2.

    PubMed

    Antushevich, Hanna; Krawczynska, Agata; Kapica, Malgorzata; Herman, Andrzej Przemyslaw; Zabielski, Romuald

    2014-01-01

    Apelin is a regulatory peptide, identified as an endogenous ligand of the Apelin receptor (APJ). Both the apelin and the APJ were detected in brain, lung, heart, mammary gland, kidney, placenta, adipose tissues and the gastrointestinal tract. Apelin is considered an important regulatory gut peptide with a potential physiological role in gastrointestinal cytoprotection, regulation of food intake and drinking behaviour. The aim of the present study was to assess the effect of the apelin on mitosis, apoptosis and the expression of DNA repair enzyme (OGG 1/2), and APJ receptor in intestinal cell lines: rat crypt (IEC-6) and human enterocyte model (Caco-2). The cell cultures were incubated with the apelin-12 (10-8 M) for 4, 6, 12, 24 and 48 h and the apoptosis (caspase 3), mitosis (Ki-67) and DNA repair enzyme (OGG1/2) markers were studied by Real-Time qRT-PCR and immunofluorescent methods. The results of Real-Time qRT-PCR and immunocytochemical analysis showed that the levels of mRNAs were inversely related to the expression level of corresponding proteins. Immunofluorescent studies revealed inhibitory effect of apelin-12 on apoptosis, mitosis and the expression of OGG1/2 in the intestinal crypt cell line IEC-6. However, in the enterocyte model Caco-2 cells apelin stimulated apoptosis and mitosis, and reduced OGG1/2 expression. These findings suggest that apelin may be involved in the control of epithelial cell turnover in the gastrointestinal tract.

  3. Expression of digestive enzymes and nutrient transporters in the small intestine of Eimeria acervulina-infected chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Coccidiosis is a major disease of poultry caused by the intestinal protozoa Eimeria. Eimeria acervulina mainly infects the duodenum, causing lesions in epithelial tissue. The objective of this study was to investigate the effect of E. acervulina infection on the expression of 18 nutrient transport...

  4. Effects of Dietary Supplementation with the Combination of Zeolite and Attapulgite on Growth Performance, Nutrient Digestibility, Secretion of Digestive Enzymes and Intestinal Health in Broiler Chickens

    PubMed Central

    Zhou, P.; Tan, Y. Q.; Zhang, L.; Zhou, Y. M.; Gao, F.; Zhou, G. H.

    2014-01-01

    This study was designed to investigate the effects of basal diets supplemented with a clay product consisting of zeolite and attapulgite (ZA) at 1:1 ratio on growth performance, digestibility of feed nutrients, activities of digestive enzymes in small intestine and intestinal health in broiler chickens. In experiment 1, 112 one-day-old male chickens were randomly divided into 2 groups with 8 replicates of 7 chickens each. In experiment 2, 84 one-day-old male chickens were randomly allocated into 2 groups consisting 6 replicates of 7 chickens each. The experimental diets both consisted of a maize-soybean basal control diet supplemented with 0% or 2% ZA. The diets were fed from 1 to 42 days of age. The results showed that ZA supplementation could increase body weight gain (BWG) and feed intake (FI), but had no significant effect on feed conversion ratio. The apparent digestibility values of crude protein and gross energy were significantly increased (p<0.05) by ZA from 14 to 16 d and 35 to 37 d. Dietary ZA treatment significantly increased (p<0.05) the activities of amylase, lipase and trypsin in jejunal digesta and the activities of maltase and sucrase in jejunal mucosa on days 21 and 42. The ZA supplementation also significantly increased (p<0.05) the catalase activity, reduced (p<0.05) the malondialdehyde concentration in the jejunal mucosa. In addition, a decrease of serum diamine oxidase activity and an increase (p<0.05) in concentration of secretory immunoglobulin A in jejunal mucosa were observed in birds treated with ZA on 21 and 42 days. It is concluded that ZA supplementation (2%) could partially improve the growth performance by increasing BWG and FI. This improvement was achieved through increasing the secretion of digestive enzymes, enhancing the digestibilites of nutrients, promoting intestinal health of broiler chickens. PMID:25178375

  5. Acyl-acyl carrier protein thioesterase activity from sunflower (Helianthus annuus L.) seeds.

    PubMed

    Martínez-Force, E; Cantisán, S; Serrano-Vega, M J; Garcés, R

    2000-10-01

    During sunflower (Helianthus annuus L.) seed formation there was an active period of lipid biosynthesis between 12 and 28 days after flowering (DAF). The maximum in-vitro acyl-acyl carrier protein (ACP) thioesterase activities (EC 3.1.2.14) were found at 15 DAF, preceding the largest accumulation of lipid in the seed. Data from the apparent kinetic parameters, Vmax and Km, from seeds of 15 and 30 DAF, showed that changes in acyl-ACP thioesterase activity are not only quantitative, but also qualitative, since, although the preferred substrate was always oleoyl-ACP, the affinity for palmitoyl-ACP decreased, whereas that for stearoyl-ACP increased with seed maturation. Bisubstrate assays carried out at 30 DAF seemed to indicate that the total activity found in mature seeds is due to a single enzyme with 100/75/15 affinity for oleoyl-ACP/stearoyl-ACP/ palmitoyl-ACP. In contrast, at 15 DAF, enzymatic data together with partial sequences from cDNAs indicated the presence of at least two enzymes with different properties, a FatA-like thioesterase, with a high affinity for oleoyl-ACP, plus a FatB-like enzyme, with preference for long-chain saturated fatty acids, both being expressed during the active lipid biosynthesis period. Competition assays carried out with CAS-5, a mutant with a higher content of palmitic acid in the seed oil, indicated that a modified FatA-type thioesterase is involved in the mutant phenotype.

  6. The enhancement of phase 2 enzyme activities by sodium butyrate in normal intestinal epithelial cells is associated with Nrf2 and p53.

    PubMed

    Yaku, Keisuke; Enami, Yuka; Kurajyo, Chika; Matsui-Yuasa, Isao; Konishi, Yotaro; Kojima-Yuasa, Akiko

    2012-11-01

    Dietary fiber fermentation by the colonic bacterial flora produces short-chain fatty acids, acetate, propionate and butyrate. Among them, butyrate is considered to be the major energy substrate for colonocytes and, at least in rats, seems to protect against colonic carcinogenesis. In this study, we examined the effect and the mechanisms of short-chain fatty acids on the activity of phase 2 enzymes. Sodium butyrate increased phase 2 enzyme activities in normal rat small intestine epithelial cells, Glutathione S-transferase and NAD(P)H:quinone oxidoreductase (NQO) in a dose-dependent manner(;) however, other short-chain fatty acids did not increase them. The mechanism of the induction of phase 2 enzymes with sodium butyrate sodium butyrate, but not other short-chain fatty acids was related to the increase of NF-E2-related factor 2 (Nrf2) nuclear translocation and the decrease in the levels of nuclear fraction p53. Sodium butyrate also caused enhancement of Nrf2 mRNA levels and suppression of p53 mRNA levels. Sodium butyrate enhances the activities of phase 2 enzymes via an increase in the Nrf2 protein levels in the nucleus and a decrease in the mRNA and protein levels of p53.

  7. Applying Data Mining to Classify Age by Intestinal Microbiota in 92 Healthy Men Using a Combination of Several Restriction Enzymes for T-RFLP Experiments.

    PubMed

    Kobayashi, Toshio; Osaki, Takako; Oikawa, Shinya

    2014-01-01

    The composition of the intestinal microbiota was measured following consumption of identical meals for 3 days in 92 Japanese men, and terminal restriction fragment length polymorphism (T-RFLP) was used to analyze their feces. The obtained operational taxonomic units (OTUs) and the subjects' ages were classified by using Data mining (DM) software that compared these data with continuous data and for 5 partitions for age divided at 5 years intervals between the ages of 30 and 50. The DM provided Decision trees in which the selected OTUs were closely related to the ages of the subjects. DM was also used to compare the OTUs from the T-RFLP data with seven restriction enzymes (two enzymes of 516f-BslI and 516f-HaeIII, two enzymes of 27f-MspI and 27f-AluI, three enzymes of 35f-HhaI, 35f-MspI and 35f-AluI) and their various combinations. The OTUs delivered from the five enzyme-digested partitions were analyzed to classify their age clusters. For use in future DM processing, we discussed the enzymes that were effective for accurate classification. We selected two OTUs (HA624 and HA995) that were useful for classifying the subject's ages. Depending on the 16S rRNA sequences of the OTUs, Ruminicoccus obeum clones 1-4 were present in 18 of 36 bacterial candidates in the older age group-related OTU (HA624). On the other hand, Ruminicoccus obeum clones 1-33 were present in 65 of 269 candidates in the younger age group-related OUT (HA995).

  8. A covalent adduct of MbtN, an acyl-ACP dehydrogenase from Mycobacterium tuberculosis, reveals an unusual acyl-binding pocket.

    PubMed

    Chai, Ai-Fen; Bulloch, Esther M M; Evans, Genevieve L; Lott, J Shaun; Baker, Edward N; Johnston, Jodie M

    2015-04-01

    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Access to iron in host macrophages depends on iron-chelating siderophores called mycobactins and is strongly correlated with Mtb virulence. Here, the crystal structure of an Mtb enzyme involved in mycobactin biosynthesis, MbtN, in complex with its FAD cofactor is presented at 2.30 Å resolution. The polypeptide fold of MbtN conforms to that of the acyl-CoA dehydrogenase (ACAD) family, consistent with its predicted role of introducing a double bond into the acyl chain of mycobactin. Structural comparisons and the presence of an acyl carrier protein, MbtL, in the same gene locus suggest that MbtN acts on an acyl-(acyl carrier protein) rather than an acyl-CoA. A notable feature of the crystal structure is the tubular density projecting from N(5) of FAD. This was interpreted as a covalently bound polyethylene glycol (PEG) fragment and resides in a hydrophobic pocket where the substrate acyl group is likely to bind. The pocket could accommodate an acyl chain of 14-21 C atoms, consistent with the expected length of the mycobactin acyl chain. Supporting this, steady-state kinetics show that MbtN has ACAD activity, preferring acyl chains of at least 16 C atoms. The acyl-binding pocket adopts a different orientation (relative to the FAD) to other structurally characterized ACADs. This difference may be correlated with the apparent ability of MbtN to catalyse the formation of an unusual cis double bond in the mycobactin acyl chain.

  9. Effect of dietary lysine on growth, intestinal enzymes activities and antioxidant status of sub-adult grass carp (Ctenopharyngodon idella).

    PubMed

    Li, Xue-Yin; Tang, Ling; Hu, Kai; Liu, Yang; Jiang, Wei-Dan; Jiang, Jun; Wu, Pei; Chen, Gang-Fu; Li, Shu-Hong; Kuang, Sheng-Yao; Feng, Lin; Zhou, Xiao-Qiu

    2014-06-01

    The dietary lysine requirement of sub-adult grass carp (460 ± 1.5 g) was assessed by feeding diets supplemented with grade levels of lysine (6.6, 8.5, 10.8, 12.9, 15.0 and 16.7 g kg(-1) diet) for 56 days. The test diets (28% CP) contained fish meal, casein and gelatin as sources of intact protein, supplemented with crystalline amino acids. Weight gain (WG), feed intake and feed efficiency were significantly improved with increasing levels of lysine up to 12.9 g kg(-1) diet and thereafter declined (P < 0.05). Quadratic regression analysis of WG at 95% maximum response indicated lysine requirement was 10.9 g kg(-1) diet. Activities of trypsin, chymotrypsin, lipase, Na(+), K(+)-ATPase and alkaline phosphatase in intestine, creatine kinase activity in proximal and mid-intestine responded similar to WG (P < 0.05). In addition, lipid and protein oxidation decreased with increasing levels of lysine up to certain values and increased thereafter (P < 0.05); the anti-hydroxyl radical capacity, dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase (GST) activities and glutathione content were increased with increasing dietary lysine levels up to certain values in the detected tissues, except for hepatopancreatic GST. Requirement estimated on the basis of malondialdehyde content in intestine and hepatopancreas was 10.6 and 9.53 g lysine kg(-1) diet, respectively. PMID:24174167

  10. Calcium-myristoyl Tug is a new mechanism for intramolecular tuning of calcium sensitivity and target enzyme interaction for guanylyl cyclase-activating protein 1: dynamic connection between N-fatty acyl group and EF-hand controls calcium sensitivity.

    PubMed

    Peshenko, Igor V; Olshevskaya, Elena V; Lim, Sunghyuk; Ames, James B; Dizhoor, Alexander M

    2012-04-20

    Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca(2+) sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca(2+) sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca(2+) binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca(2+)-bound and Mg(2+)-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca(2+) sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca(2+) binding to EF-hand 4 and Ca(2+) sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu(80) and Val(180)) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or "tug") between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca(2+) sensitivity.

  11. Interplay between the chalcone cardamonin and selenium in the biosynthesis of Nrf2-regulated antioxidant enzymes in intestinal Caco-2 cells.

    PubMed

    De Spirt, Silke; Eckers, Anna; Wehrend, Carina; Micoogullari, Mustafa; Sies, Helmut; Stahl, Wilhelm; Steinbrenner, Holger

    2016-02-01

    Selenoenzymes and nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated phase II enzymes comprise key components of the cellular redox and antioxidant systems, which show multiple interrelations. Deficiency of the micronutrient selenium (Se) and impaired biosynthesis of selenoproteins have been reported to result in induction of Nrf2 target genes. Conversely, transcription of the selenoenzymes glutathione peroxidase 2 (GPx2) and thioredoxin reductase 1 (TrxR1) is up-regulated upon Nrf2 activation. Here, we have studied the interplay between Se and the secondary plant metabolite cardamonin, an Nrf2-activating chalcone, in the regulation of Nrf2-controlled antioxidant enzymes. Se-deficient and Se-repleted (sodium selenite-supplemented) human intestinal Caco-2 cells were exposed to cardamonin. Uptake of cardamonin by the Caco-2 cells was independent of their Se status. Cardamonin strongly induced gene expression of GPx2 and TrxR1. However, cardamonin treatment did not result in elevated GPx or TrxR activity and protein levels, possibly relating to a concomitant down-regulation of O-phosphoseryl-tRNA(Sec) kinase (PSTK), an enzyme involved in translation of selenoprotein mRNAs. On the other hand, induction of the Nrf2-regulated enzyme heme oxygenase 1 (HO-1) by cardamonin was diminished in Se-replete compared to Se-deficient cells. Our findings suggest that cardamonin interferes with the biosynthesis of Nrf2-regulated selenoenzymes, in contrast to the Nrf2-activating isothiocyanate compound sulforaphane, which has been shown earlier to synergize with Se-mediated cytoprotection. Conversely, the cellular Se status apparently affects the cardamonin-mediated induction of non-selenoprotein antioxidant enzymes such as HO-1. PMID:26698667

  12. The ɛ-Amino Group of Protein Lysine Residues Is Highly Susceptible to Nonenzymatic Acylation by Several Physiological Acyl-CoA Thioesters.

    PubMed

    Simic, Zeljko; Weiwad, Matthias; Schierhorn, Angelika; Steegborn, Clemens; Schutkowski, Mike

    2015-11-01

    Mitochondrial enzymes implicated in the pathophysiology of diabetes, cancer, and metabolic syndrome are highly regulated by acetylation. However, mitochondrial acetyltransferases have not been identified. Here, we show that acetylation and also other acylations are spontaneous processes that depend on pH value, acyl-CoA concentration and the chemical nature of the acyl residue. In the case of a peptide derived from carbamoyl phosphate synthetase 1, the rates of succinylation and glutarylation were up to 150 times than for acetylation. These results were confirmed by using the protein substrate cyclophilin A (CypA). Deacylation experiments revealed that SIRT3 exhibits deacetylase activity but is not able to remove any of the succinyl groups from CypA, whereas SIRT5 is an effective protein desuccinylase. Thus, the acylation landscape on lysine residues might largely depend on the enzymatic activity of specific sirtuins, and the availability and reactivity of acyl-CoA compounds. PMID:26382620

  13. The functional size of acyl-coenzyme A (CoA):cholesterol acyltransferase and acyl-CoA hydrolase as determined by radiation inactivation

    SciTech Connect

    Billheimer, J.T.; Cromley, D.A.; Kempner, E.S. )

    1990-05-25

    Frozen rat liver microsomes and rough endoplasmic reticulum were irradiated with high energy electrons. The surviving enzymatic activity of acyl-CoA:cholesterol acyltransferase and activity for esterification of 25-hydroxycholesterol decreased as a simple exponential function of radiation exposure, leading to a target size of 170-180 kDa. The loss of acyl-CoA hydrolase activity with a radiation dose was complex and resolved as a 45-kDa enzyme associated with a large inhibitor. It is interpreted that acyl-CoA hydrolase is the acyl-CoA-binding component and the inhibitor is the cholesterol-binding component of acyl-CoA:cholesterol acyltransferase.

  14. Mammalian long-chain acyl-CoA synthetases.

    PubMed

    Soupene, Eric; Kuypers, Frans A

    2008-05-01

    Acyl-CoA synthetase enzymes are essential for de novo lipid synthesis, fatty acid catabolism, and remodeling of membranes. Activation of fatty acids requires a two-step reaction catalyzed by these enzymes. In the first step, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with CoA to produce the activated acyl-CoA. The release of AMP in this reaction defines the superfamily of AMP-forming enzymes. The length of the carbon chain of the fatty acid species defines the substrate specificity for the different acyl-CoA synthetases (ACS). On this basis, five sub-families of ACS have been characterized. The purpose of this review is to report on the large family of mammalian long-chain acyl-CoA synthetases (ACSL), which activate fatty acids with chain lengths of 12 to 20 carbon atoms. Five genes and several isoforms generated by alternative splicing have been identified and limited information is available on their localization. The structure of these membrane proteins has not been solved for the mammalian ACSLs but homology to a bacterial form, whose structure has been determined, points at specific structural features that are important for these enzymes across species. The bacterial form acts as a dimer and has a conserved short motif, called the fatty acid Gate domain, that seems to determine substrate specificity. We will discuss the characterization and identification of the different spliced isoforms, draw attention to the inconsistencies and errors in their annotations, and their cellular localizations. These membrane proteins act on membrane-bound substrates probably as homo- and as heterodimer complexes but have often been expressed as single recombinant isoforms, apparently purified as monomers and tested in Triton X-100 micelles. We will argue that such studies have failed to provide an accurate assessment of the activity and of the distinct function of these enzymes in mammalian cells.

  15. Intestinal α-glucosidase and some pancreatic enzymes inhibitory effect of hydroalcholic extract of Moringa stenopetala leaves

    PubMed Central

    2014-01-01

    Background Moringa stenopetala has been used in traditional health systems to treat diabetes mellitus. One of the successful methods to prevent of the onset of diabetes is to control postprandial hyperglycemia by the inhibition of α-glucosidase and pancreatic α-amylase activities, resulting in the aggressive delay of the carbohydrate digestion of absorbable monosaccharides. The aim of the present study is to investigate the effect of the extract of the leaves of Moringa stenopetala on α-glucosidase, pancreatic α-amylase, pancreatic lipase, and pancreatic cholesterol esterase activities, and, therefore find out the relevance of the plant in controlling blood sugar and lipid levels. Methods The dried leaves of Moringa stenopetala were extracted with hydroalcoholic solvent and dried using rotary vapor under reduced pressure. The dried extracts were determined for the total phenolic compounds, flavonoid content and condensed tannins content by using Folin-Ciocateu’s reagent, AlCl3 and vanillin assay, respectively. The dried extract of plant-based food was further quantified with respect to intestinal α-glucosidase (maltase and sucrase) inhibition and pancreatic α-amylase inhibition by glucose oxidase method and dinitrosalicylic (DNS) reagent, respectively. Results The phytochemical analysis indicated that flavonoid, total phenolic, and condensed tannin contents in the extract were 71.73 ± 2.48 mg quercetin equivalent/g of crude extract, 79.81 ± 2.85 mg of gallic acid equivalent/g of crude extract, 8.82 ± 0.77 mg catechin equivalent/g of crude extract, respectively. The extract inhibited intestinal sucrase more than intestinal maltase with IC50 value of 1.47 ± 0.19 mg/ml. It also slightly inhibited pancreatic α-amylase, pancreatic lipase and pancreatic cholesterol esterase. Conclusion The result demonstrated the beneficial biochemical effects of Moringa stenopetala by inhibiting intestinal α-glucosidase, pancreatic cholesterol esterase

  16. Comparison among Different Gilthead Sea Bream (Sparus aurata) Farming Systems: Activity of Intestinal and Hepatic Enzymes and 13C-NMR Analysis of Lipids

    PubMed Central

    Coco, Laura Del; Papadia, Paride; Pascali, Sandra A. De; Bressani, Giorgia; Storelli, Carlo; Zonno, Vincenzo; Fanizzi, Francesco Paolo

    2009-01-01

    In order to evaluate differences in general health and nutritional values of gilthead sea bream (Sparus aurata), the effects of semi-intensive, land-based tanks and sea-cages intensive rearing systems were investigated, and results compared with captured wild fish. The physiological state was determined by measuring the activity of three different intestinal digestive enzymes: alkaline phosphatase (ALP), leucine aminopeptidase (LAP) and maltase; and the activity of the hepatic ALP. Also, the hepatic content in protein, cholesterol, and lipid were assessed. 13C-NMR analysis for qualitative and quantitative characterization of the lipid fraction extracted from fish muscles for semi-intensive and land based tanks intensive systems was performed. The lipid fraction composition showed small but significant differences in the monounsaturated/saturated fatty acid ratio, with the semi-intensive characterized by higher monounsaturated and lower saturated fatty acid content with respect to land based tanks intensive rearing system. PMID:22253985

  17. Quantum chemical study of penicillin: Reactions after acylation

    NASA Astrophysics Data System (ADS)

    Li, Rui; Feng, Dacheng; Zhu, Feng

    The density functional theory methods were used on the model molecules of penicillin to determine the possible reactions after their acylation on ?-lactamase, and the results were compared with sulbactam we have studied. The results show that, the acylated-enzyme tetrahedral intermediate can evolves with opening of ?-lactam ring as well as the thiazole ring; the thiazole ring-open products may be formed via ?-lactam ring-open product or from tetrahedral intermediate directly. Those products, in imine or enamine form, can tautomerize via hydrogen migration. In virtue of the water-assisted, their energy barriers are obviously reduced.

  18. Diverse Activities of Histone Acylations Connect Metabolism to Chromatin Function.

    PubMed

    Dutta, Arnob; Abmayr, Susan M; Workman, Jerry L

    2016-08-18

    Modifications of histones play important roles in balancing transcriptional output. The discovery of acyl marks, besides histone acetylation, has added to the functional diversity of histone modifications. Since all modifications use metabolic intermediates as substrates for chromatin-modifying enzymes, the prevalent landscape of histone modifications in any cell type is a snapshot of its metabolic status. Here, we review some of the current findings of how differential use of histone acylations regulates gene expression as response to metabolic changes and differentiation programs. PMID:27540855

  19. Atorvastatin induces bile acid-synthetic enzyme Cyp7a1 by suppressing FXR signaling in both liver and intestine in mice.

    PubMed

    Fu, Zidong Donna; Cui, Julia Yue; Klaassen, Curtis D

    2014-12-01

    Statins are effective cholesterol-lowering drugs to treat CVDs. Bile acids (BAs), the end products of cholesterol metabolism in the liver, are important nutrient and energy regulators. The present study aims to investigate how statins affect BA homeostasis in the enterohepatic circulation. Male C57BL/6 mice were treated with atorvastatin (100 mg/kg/day po) for 1 week, followed by BA profiling by ultra-performance LC-MS/MS. Atorvastatin decreased BA pool size, mainly due to less BA in the intestine. Surprisingly, atorvastatin did not alter total BAs in the serum or liver. Atorvastatin increased the ratio of 12α-OH/non12α-OH BAs. Atorvastatin increased the mRNAs of the BA-synthetic enzymes cholesterol 7α-hydroxylase (Cyp7a1) (over 10-fold) and cytochrome P450 27a1, the BA uptake transporters Na⁺/taurocholate cotransporting polypeptide and organic anion transporting polypeptide 1b2, and the efflux transporter multidrug resistance-associated protein 2 in the liver. Noticeably, atorvastatin suppressed the expression of BA nuclear receptor farnesoid X receptor (FXR) target genes, namely small heterodimer partner (liver) and fibroblast growth factor 15 (ileum). Furthermore, atorvastatin increased the mRNAs of the organic cation uptake transporter 1 and cholesterol efflux transporters Abcg5 and Abcg8 in the liver. The increased expression of BA-synthetic enzymes and BA transporters appear to be a compensatory response to maintain BA homeostasis after atorvastatin treatment. The Cyp7a1 induction by atorvastatin appears to be due to suppressed FXR signaling in both the liver and intestine.

  20. Atorvastatin induces bile acid-synthetic enzyme Cyp7a1 by suppressing FXR signaling in both liver and intestine in mice[S

    PubMed Central

    Fu, Zidong Donna; Cui, Julia Yue; Klaassen, Curtis D.

    2014-01-01

    Statins are effective cholesterol-lowering drugs to treat CVDs. Bile acids (BAs), the end products of cholesterol metabolism in the liver, are important nutrient and energy regulators. The present study aims to investigate how statins affect BA homeostasis in the enterohepatic circulation. Male C57BL/6 mice were treated with atorvastatin (100 mg/kg/day po) for 1 week, followed by BA profiling by ultra-performance LC-MS/MS. Atorvastatin decreased BA pool size, mainly due to less BA in the intestine. Surprisingly, atorvastatin did not alter total BAs in the serum or liver. Atorvastatin increased the ratio of 12α-OH/non12α-OH BAs. Atorvastatin increased the mRNAs of the BA-synthetic enzymes cholesterol 7α-hydroxylase (Cyp7a1) (over 10-fold) and cytochrome P450 27a1, the BA uptake transporters Na+/taurocholate cotransporting polypeptide and organic anion transporting polypeptide 1b2, and the efflux transporter multidrug resistance-associated protein 2 in the liver. Noticeably, atorvastatin suppressed the expression of BA nuclear receptor farnesoid X receptor (FXR) target genes, namely small heterodimer partner (liver) and fibroblast growth factor 15 (ileum). Furthermore, atorvastatin increased the mRNAs of the organic cation uptake transporter 1 and cholesterol efflux transporters Abcg5 and Abcg8 in the liver. The increased expression of BA-synthetic enzymes and BA transporters appear to be a compensatory response to maintain BA homeostasis after atorvastatin treatment. The Cyp7a1 induction by atorvastatin appears to be due to suppressed FXR signaling in both the liver and intestine. PMID:25278499

  1. Effect of γ-aminobutyric acid on digestive enzymes, absorption function, and immune function of intestinal mucosa in heat-stressed chicken.

    PubMed

    Chen, Z; Xie, J; Wang, B; Tang, J

    2014-10-01

    To explore the effect of dietary γ-aminobutyric acid (GABA) on digestive enzyme activity, absorption function and immune function of intestinal mucosa in heat-stressed Wenchang chicken were studied. One-day-old male Wenchang chickens were randomly divided into a control group (CK), heat stress group (HS), and GABA+HS group. The chickens from the GABA+HS group were administered with 0.2 mL of GABA solution daily. Chickens from HS and GABA+HS groups were subjected to heat stress treatment at 40 ± 0.5°C for 2 h during 1300 to 1500 h every day. Blood was drawn and 0.5 cm-long duodenum, jejunum, and ileum were collected from the chickens on d 3, 5, 7, 9, 12, and 15. Results showed that the activity of Ca²⁺-Mg²⁺-adenosine triphosphatase (ATPase), Na⁺-K⁺-ATPase, maltase, sucrase, and alkaline phosphatase, the contents of secretory IgA, glutathione, and d-xylose, and the number of lymphocytes in HS group were significantly lower than those in the CK group. Among them, some were rescued after the treatment of GABA as the time extension. For maltase, d-xylose, alkaline phosphatase, and Na⁺-K⁺-ATPase, it required 5 to 7 d for achieving the significant effect. For sucrase, 12 d for the alleviation effect was required. In the case of other parameters, no alleviation was observed during the whole period of the study. We have concluded that HS can inhibit the activity of digestive enzymes and reduce absorption and immune functions of intestinal mucosa. γ-Aminobutyric acid can effectively alleviate these inhibitory effects.

  2. [Activity of oxidation-reduction enzymes in endotheliocytes of the intestinal hemomicrocirculatory bed under normal conditions and in portal hypertension].

    PubMed

    Gaĭvoronskiĭ, I V; Tikhonova, L P; Chepur, S V; Nichiporuk, G I

    1997-01-01

    An original quantitative examination of oxidation-reduction enzymes activity in endotheliocytes of hemomicroclrculatory vessels of jejunum and rectum submucosal base in normal state and in portal hypertension was performed by the authors. Comparative analysis of the activity of the enzymes studied revealed different metabolic processes intensity in these organs, dependent on current hemodynamic conditions. Cytochemical changes in hemomicrocirculatory bed are consistent with structural reorganizations that arise in the wall of vessels studied, consist of several phases and may be used as an assessment criterion for defining the portal hypertension stage.

  3. 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…

  4. Acylation of Streptomyces type II polyketide synthase acyl carrier proteins.

    PubMed

    Crosby, J; Byrom, K J; Hitchman, T S; Cox, R J; Crump, M P; Findlow, I S; Bibb, M J; Simpson, T J

    1998-08-14

    Acyl derivatives of type II PKS ACPs are required for in vitro studies of polyketide biosynthesis. The presence of an exposed cysteine residue prevented specific chemical acylation of the phosphopantetheine thiol of the actinorhodin PKS holo ACP. Acylation studies were further complicated by intramolecular disulphide formation between cysteine 17 and the phosphopantetheine. The presence of this intramolecular disulphide was confirmed by tryptic digestion of the ACP followed by ESMS analysis of the fragments. An act Cys17Ser ACP was engineered by site-directed mutagenesis. S-Acyl adducts of act C17S, oxytetracycline and griseusin holo ACPs were rapidly formed by reaction with hexanoyl, 5-ketohexanoyl and protected acetoacetyl imidazolides. Comparisons with type 11 FAS ACPs were made.

  5. The presence of acyl-CoA hydrolase in rat brown-adipose-tissue peroxisomes.

    PubMed

    Alexson, S E; Osmundsen, H; Berge, R K

    1989-08-15

    The subcellular distribution of acyl-CoA hydrolase was studied in rat brown adipose tissue, with special emphasis on possible peroxisomal localization. Subcellular fractionation by sucrose-density-gradient centrifugation, followed by measurement of short-chain (propionyl-CoA) acyl-CoA hydrolase in the presence of NADH, resulted in two peaks of activity in the gradient: one peak corresponded to the distribution of cytochrome oxidase (mitochondrial marker enzyme), and another peak of activity coincided with the peroxisomal marker enzyme catalase. The distribution of the NADH-inhibited short-chain hydrolase activity fully resembled that of cytochrome oxidase. The substrate-specificity curve of the peroxisomal acyl-CoA hydrolase activity indicated the presence of a single enzyme exhibiting a broad substrate specificity, with maximal activity towards fatty acids with chain lengths of 3-12 carbon atoms. The mitochondrial acyl-CoA hydrolase substrate specificity, in contrast, indicated the presence of at least two acyl-CoA hydrolases (of short- and medium-chain-length specificity). The peroxisomal acyl-CoA hydrolase activity was inhibited by CoA at low (microM) concentrations and by ATP at high concentrations (greater than 0.8 mM). In contrast with the mitochondrial short-chain hydrolase, the peroxisomal acyl-CoA hydrolase activity was not inhibited by NADH. PMID:2573347

  6. A novel 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase from Advenella mimigardefordensis strain DPN7T acting as a key enzyme during catabolism of 3,3'-dithiodipropionic acid is a member of the acyl-CoA dehydrogenase superfamily.

    PubMed

    Schürmann, Marc; Deters, Anika; Wübbeler, Jan Hendrik; Steinbüchel, Alexander

    2013-04-01

    3-Sulfinopropionyl coenzyme A (3SP-CoA) desulfinase (AcdDPN7) is a new desulfinase that catalyzes the sulfur abstraction from 3SP-CoA in the betaproteobacterium Advenella mimigardefordensis strain DPN7(T). During investigation of a Tn5::mob-induced mutant defective in growth on 3,3'-dithiodipropionate (DTDP) and also 3-sulfinopropionate (3SP), the transposon insertion was mapped to an open reading frame with the highest homology to an acyl-CoA dehydrogenase (Acd) from Burkholderia phenoliruptrix strain BR3459a (83% identical and 91% similar amino acids). An A. mimigardefordensis Δacd mutant was generated and verified the observed phenotype of the Tn5::mob-induced mutant. For enzymatic studies, AcdDPN7 was heterologously expressed in Escherichia coli BL21(DE3)/pLysS by using pET23a::acdDPN7. The purified protein is yellow and contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor, as verified by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) analyses. Size-exclusion chromatography revealed a native molecular mass of about 173 kDa, indicating a homotetrameric structure (theoretically 179 kDa), which is in accordance with other members of the acyl-CoA dehydrogenase superfamily. In vitro assays unequivocally demonstrated that the purified enzyme converted 3SP-CoA into propionyl-CoA and sulfite (SO3(2-)). Kinetic studies of AcdDPN7 revealed a Vmax of 4.19 μmol min(-1) mg(-1), an apparent Km of 0.013 mM, and a kcat/Km of 240.8 s(-1) mM(-1) for 3SP-CoA. However, AcdDPN7 is unable to perform a dehydrogenation, which is the usual reaction catalyzed by members of the acyl-CoA dehydrogenase superfamily. Comparison to other known desulfinases showed a comparably high catalytic efficiency of AcdDPN7 and indicated a novel reaction mechanism. Hence, AcdDPN7 encodes a new desulfinase based on an acyl-CoA dehydrogenase (EC 1.3.8.x) scaffold. Concomitantly, we identified the gene product that is responsible for the

  7. A Novel 3-Sulfinopropionyl Coenzyme A (3SP-CoA) Desulfinase from Advenella mimigardefordensis Strain DPN7T Acting as a Key Enzyme during Catabolism of 3,3′-Dithiodipropionic Acid Is a Member of the Acyl-CoA Dehydrogenase Superfamily

    PubMed Central

    Schürmann, Marc; Deters, Anika; Wübbeler, Jan Hendrik

    2013-01-01

    3-Sulfinopropionyl coenzyme A (3SP-CoA) desulfinase (AcdDPN7) is a new desulfinase that catalyzes the sulfur abstraction from 3SP-CoA in the betaproteobacterium Advenella mimigardefordensis strain DPN7T. During investigation of a Tn5::mob-induced mutant defective in growth on 3,3′-dithiodipropionate (DTDP) and also 3-sulfinopropionate (3SP), the transposon insertion was mapped to an open reading frame with the highest homology to an acyl-CoA dehydrogenase (Acd) from Burkholderia phenoliruptrix strain BR3459a (83% identical and 91% similar amino acids). An A. mimigardefordensis Δacd mutant was generated and verified the observed phenotype of the Tn5::mob-induced mutant. For enzymatic studies, AcdDPN7 was heterologously expressed in Escherichia coli BL21(DE3)/pLysS by using pET23a::acdDPN7. The purified protein is yellow and contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor, as verified by high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC-ESI-MS) analyses. Size-exclusion chromatography revealed a native molecular mass of about 173 kDa, indicating a homotetrameric structure (theoretically 179 kDa), which is in accordance with other members of the acyl-CoA dehydrogenase superfamily. In vitro assays unequivocally demonstrated that the purified enzyme converted 3SP-CoA into propionyl-CoA and sulfite (SO32−). Kinetic studies of AcdDPN7 revealed a Vmax of 4.19 μmol min−1 mg−1, an apparent Km of 0.013 mM, and a kcat/Km of 240.8 s−1 mM−1 for 3SP-CoA. However, AcdDPN7 is unable to perform a dehydrogenation, which is the usual reaction catalyzed by members of the acyl-CoA dehydrogenase superfamily. Comparison to other known desulfinases showed a comparably high catalytic efficiency of AcdDPN7 and indicated a novel reaction mechanism. Hence, AcdDPN7 encodes a new desulfinase based on an acyl-CoA dehydrogenase (EC 1.3.8.x) scaffold. Concomitantly, we identified the gene product that is responsible for

  8. Regioselective self-acylating cyclodextrins in organic solvent

    PubMed Central

    Cho, Eunae; Yun, Deokgyu; Jeong, Daham; Im, Jieun; Kim, Hyunki; Dindulkar, Someshwar D.; Choi, Youngjin; Jung, Seunho

    2016-01-01

    Amphiphilic cyclodextrins have been synthesized with self-acylating reaction using vinyl esters in dimethylformamide. In the present study no base, catalyst, or enzyme was used, and the structural analyses using thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry show that the cyclodextrin is substituted preferentially by one acyl moiety at the C2 position of the glucose unit, suggesting that cyclodextrin functions as a regioselective catalytic carbohydrate in organic solvent. In the self-acylation, the most acidic OH group at the 2-position and the inclusion complexing ability of cyclodextrin were considered to be significant. The substrate preference was also observed in favor of the long-chain acyl group, which could be attributed to the inclusion ability of cyclodextrin cavity. Furthermore, using the model amphiphilic building block, 2-O-mono-lauryl β-cyclodextrin, the self-organized supramolecular architecture with nano-vesicular morphology in water was investigated by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The cavity-type nano-assembled vesicle and the novel synthetic methods for the preparation of mono-acylated cyclodextrin should be of great interest with regard to drug/gene delivery systems, functional surfactants, and carbohydrate derivatization methods. PMID:27020946

  9. Regioselective self-acylating cyclodextrins in organic solvent

    NASA Astrophysics Data System (ADS)

    Cho, Eunae; Yun, Deokgyu; Jeong, Daham; Im, Jieun; Kim, Hyunki; Dindulkar, Someshwar D.; Choi, Youngjin; Jung, Seunho

    2016-03-01

    Amphiphilic cyclodextrins have been synthesized with self-acylating reaction using vinyl esters in dimethylformamide. In the present study no base, catalyst, or enzyme was used, and the structural analyses using thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry show that the cyclodextrin is substituted preferentially by one acyl moiety at the C2 position of the glucose unit, suggesting that cyclodextrin functions as a regioselective catalytic carbohydrate in organic solvent. In the self-acylation, the most acidic OH group at the 2-position and the inclusion complexing ability of cyclodextrin were considered to be significant. The substrate preference was also observed in favor of the long-chain acyl group, which could be attributed to the inclusion ability of cyclodextrin cavity. Furthermore, using the model amphiphilic building block, 2-O-mono-lauryl β-cyclodextrin, the self-organized supramolecular architecture with nano-vesicular morphology in water was investigated by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The cavity-type nano-assembled vesicle and the novel synthetic methods for the preparation of mono-acylated cyclodextrin should be of great interest with regard to drug/gene delivery systems, functional surfactants, and carbohydrate derivatization methods.

  10. Regioselective self-acylating cyclodextrins in organic solvent.

    PubMed

    Cho, Eunae; Yun, Deokgyu; Jeong, Daham; Im, Jieun; Kim, Hyunki; Dindulkar, Someshwar D; Choi, Youngjin; Jung, Seunho

    2016-01-01

    Amphiphilic cyclodextrins have been synthesized with self-acylating reaction using vinyl esters in dimethylformamide. In the present study no base, catalyst, or enzyme was used, and the structural analyses using thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry show that the cyclodextrin is substituted preferentially by one acyl moiety at the C2 position of the glucose unit, suggesting that cyclodextrin functions as a regioselective catalytic carbohydrate in organic solvent. In the self-acylation, the most acidic OH group at the 2-position and the inclusion complexing ability of cyclodextrin were considered to be significant. The substrate preference was also observed in favor of the long-chain acyl group, which could be attributed to the inclusion ability of cyclodextrin cavity. Furthermore, using the model amphiphilic building block, 2-O-mono-lauryl β-cyclodextrin, the self-organized supramolecular architecture with nano-vesicular morphology in water was investigated by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The cavity-type nano-assembled vesicle and the novel synthetic methods for the preparation of mono-acylated cyclodextrin should be of great interest with regard to drug/gene delivery systems, functional surfactants, and carbohydrate derivatization methods. PMID:27020946

  11. Utilization of peptide carrier system to improve intestinal absorption: targeting prolidase as a prodrug-converting enzyme

    NASA Technical Reports Server (NTRS)

    Bai, J. P.; Hu, M.; Subramanian, P.; Mosberg, H. I.; Amidon, G. L.

    1992-01-01

    The feasibility of targeting prolidase as a peptide prodrug-converting enzyme has been examined. The enzymatic hydrolysis by prolidase of substrates for the peptide transporter L-alpha-methyldopa-pro and several dipeptide analogues without an N-terminal alpha-amino group (phenylpropionylproline, phenylacetylproline, N-benzoylproline, and N-acetylproline) was investigated. The Michaelis-Menten parameters Km and Vmax for L-alpha-methyldopa-pro are 0.09 +/- 0.02 mM and 3.98 +/- 0.25 mumol/min/mg protein, respectively. However, no hydrolysis of the dipeptide analogues without an N-terminal alpha-amino group is observed, suggesting that an N-terminal alpha-amino group is required for prolidase activity. These results demonstrate that prolidase may serve as a prodrug-converting enzyme for the dipeptide-type prodrugs, utilizing the peptide carrier for transport of prodrugs into the mucosal cells and prolidase, a cytosolic enzyme, to release the drug. However, a free alpha-amino group appears to be necessary for prolidase hydrolysis.

  12. Determination Trial of Nondigestible Oligosaccharide in Processed Foods by Improved AOAC Method 2009.01 Using Porcine Small Intestinal Enzyme.

    PubMed

    Tanabe, Kenichi; Nakamura, Sadako; Omagari, Katsuhisa; Oku, Tsuneyuki

    2015-06-24

    We have previously shown that the Association of Official Analytical Chemists' (AOAC) methods 2001.03 and 2009.01 were not able to measure accurately nondigestible oligosaccharide because they are incapable of hydrolyzing digestible oligosaccharide, leading to overestimation of nondigestible oligosaccharide. Subsequently, we have proposed improved AOAC methods 2001.03 and 2009.01 using porcine small intestinal disaccharidases instead of amyloglucosidase. In the present study, we tried to determine nondigestible oligosaccharide in marketed processed foods using the improved AOAC method (improved method), and the results were compared with those by AOAC method 2009.01. In the improved method, the percentages of recovery of fructooligosaccharide, galactooligosaccharide, and raffinose to the label of processed food were 103.0, 89.9, and 102.1%, respectively. However, the AOAC method 2009.01 overestimated >30% of the quantity of nondigestible oligosaccharide in processed foods, because the margin of error was accepted ±20% on the contents of nondigestible oligosaccharides in processed foods for Japanese nutrition labeling, the improved method thus provided accurate quantification of nondigestible oligosaccharides in processed food and allows a comprehensive determination of nondigestible oligosaccharides.

  13. Discovery of amide (peptide) bond synthetic activity in Acyl-CoA synthetase.

    PubMed

    Abe, Tomoko; Hashimoto, Yoshiteru; Hosaka, Hideaki; Tomita-Yokotani, Kaori; Kobayashi, Michihiko

    2008-04-25

    Acyl-CoA synthetase, which is one of the acid-thiol ligases (EC 6.2.1), plays key roles in metabolic and regulatory processes. This enzyme forms a carbon-sulfur bond in the presence of ATP and Mg(2+), yielding acyl-CoA thioesters from the corresponding free acids and CoA. This enzyme belongs to the superfamily of adenylate-forming enzymes, whose three-dimensional structures are analogous to one another. We here discovered a new reaction while studying the short-chain acyl-CoA synthetase that we recently reported (Hashimoto, Y., Hosaka, H., Oinuma, K., Goda, M., Higashibata, H., and Kobayashi, M. (2005) J. Biol. Chem. 280, 8660-8667). When l-cysteine was used as a substrate instead of CoA, N-acyl-l-cysteine was surprisingly detected as a reaction product. This finding demonstrated that the enzyme formed a carbon-nitrogen bond (EC 6.3.1 acid-ammonia (or amide) ligase (amide synthase); EC 6.3.2 acid-amino acid ligase (peptide synthase)) comprising the amino group of the cysteine and the carboxyl group of the acid. N-Acyl-d-cysteine, N-acyl-dl-homocysteine, and N-acyl-l-cysteine methyl ester were also synthesized from the corresponding cysteine analog substrates by the enzyme. Furthermore, this unexpected enzyme activity was also observed for acetyl-CoA synthetase and firefly luciferase, indicating the generality of the new reaction in the superfamily of adenylate-forming enzymes.

  14. Permeation and metabolism of a series of novel lipophilic ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain, in a human living skin equivalent model.

    PubMed

    Tai, Akihiro; Goto, Satomi; Ishiguro, Yutaka; Suzuki, Kazuko; Nitoda, Teruhiko; Yamamoto, Itaru

    2004-02-01

    A series of novel lipophilic vitamin C derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids possessing a branched-acyl chain of varying length from C(8) to C(16) (6-bAcyl-AA-2G), were evaluated as topical prodrugs of ascorbic acid (AA) with transdermal activity in a human living skin equivalent model. The permeability of 6-bAcyl-AA-2G was compared with those of the derivatives having a straight-acyl chain (6-sAcyl-AA-2G). Out of 10 derivatives of 6-sAcyl-AA-2G and 6-bAcyl-AA-2G, 6-sDode-AA-2G and 6-bDode-AA-2G exhibited most excellent permeability in this model. Measurement of the metabolites permeated from the skin model suggested that 6-bDode-AA-2G was mainly hydrolyzed via 6-O-acyl AA to AA by tissue enzymes, while 6-sDode-AA-2G was hydrolyzed via 2-O-alpha-D-glucopyranosyl-L-ascorbic acid to AA. The former metabolic pathway seems to be advantageous for a readily available source of AA, because 6-O-acyl AA, as well as AA, is able to show vitamin C activity.

  15. Fatty acylation of proteins: The long and the short of it.

    PubMed

    Resh, Marilyn D

    2016-07-01

    Long, short and medium chain fatty acids are covalently attached to hundreds of proteins. Each fatty acid confers distinct biochemical properties, enabling fatty acylation to regulate intracellular trafficking, subcellular localization, protein-protein and protein-lipid interactions. Myristate and palmitate represent the most common fatty acid modifying groups. New insights into how fatty acylation reactions are catalyzed, and how fatty acylation regulates protein structure and function continue to emerge. Myristate is typically linked to an N-terminal glycine, but recent studies reveal that lysines can also be myristoylated. Enzymes that remove N-terminal myristoyl-glycine or myristate from lysines have now been identified. DHHC proteins catalyze S-palmitoylation, but the mechanisms that regulate substrate recognition by individual DHHC family members remain to be determined. New studies continue to reveal thioesterases that remove palmitate from S-acylated proteins. Another area of rapid expansion is fatty acylation of the secreted proteins hedgehog, Wnt and Ghrelin, by Hhat, Porcupine and GOAT, respectively. Understanding how these membrane bound O-acyl transferases recognize their protein and fatty acyl CoA substrates is an active area of investigation, and is punctuated by the finding that these enzymes are potential drug targets in human diseases. PMID:27233110

  16. Characterization of a Bifunctional Archaeal Acyl Coenzyme A Carboxylase

    PubMed Central

    Chuakrut, Songkran; Arai, Hiroyuki; Ishii, Masaharu; Igarashi, Yasuo

    2003-01-01

    Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from Acidianus brierleyi. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an α4β4γ4 subunit structure. The optimum temperature for the enzyme was 60 to 70°C, and the optimum pH was around 6.4 to 6.9. Interestingly, the purified enzyme also had propionyl-CoA carboxylase activity. The apparent Km for acetyl-CoA was 0.17 ± 0.03 mM, with a Vmax of 43.3 ± 2.8 U mg−1, and the Km for propionyl-CoA was 0.10 ± 0.008 mM, with a Vmax of 40.8 ± 1.0 U mg−1. This result showed that A. brierleyi acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. Both enzymatic activities were inhibited by malonyl-CoA, methymalonyl-CoA, succinyl-CoA, or CoA but not by palmitoyl-CoA. The gene encoding acyl-CoA carboxylase was cloned and characterized. Homology searches of the deduced amino acid sequences of the 62-, 59-, and 20-kDa subunits indicated the presence of functional domains for carboxyltransferase, biotin carboxylase, and biotin carboxyl carrier protein, respectively. Amino acid sequence alignment of acetyl-CoA carboxylases revealed that archaeal acyl-CoA carboxylases are closer to those of Bacteria than to those of Eucarya. The substrate-binding motifs of the enzymes are highly conserved among the three domains. The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit. PMID:12533469

  17. Vasoactive intestinal peptide-induced expression of cytochrome P450 cholesterol side-chain cleavage and 17 alpha-hydroxylase enzyme activity in hen granulosa cells.

    PubMed

    Johnson, A L; Li, Z; Gibney, J A; Malamed, S

    1994-08-01

    Experiments were conducted to determine whether vasoactive intestinal peptide (VIP) can regulate expression of cytochrome P450 side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase (P450 17 alpha-OH) mRNA levels and enzyme activity in granulosa cells from nonhierarchal (6-8-mm) follicles. Initial studies demonstrated that immunoreactive VIP is localized within the theca (but not granulosa) layer of both resting (< 0.5-mm follicles) and 6-8-mm follicles, thus providing a potential paracrine mechanism of action for VIP. While short-term (3 h) incubation of granulosa cells with VIP (0.001-1.0 microM) failed to stimulate progesterone production from 6-8-mm follicle granulosa cells, a 4-h culture period in the presence of VIP resulted in increased cyclic AMP (cAMP) accumulation, and a 24-h culture period resulted in progesterone synthesis and increased P450scc mRNA levels; control levels of each endpoint measurement were not altered within the period observed. By contrast, culture with the growth factor transforming growth factor alpha (TGF alpha) in the presence of VIP (1 microM) prevented increases in P450scc mRNA levels and progesterone production. Similar effects of VIP and TGF alpha in the presence of VIP were demonstrated for P450 17 alpha-OH mRNA levels and enzyme activity. Finally, there was an additive effect of VIP (0.1 microM) plus recombinant human (rh) FSH (100 mIU) on the initiation of progesterone production in cultured 6-8-mm follicle granulosa cells compared to the addition of VIP or rhFSH alone.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Effects of Metabolites Produced from (-)-Epigallocatechin Gallate by Rat Intestinal Bacteria on Angiotensin I-Converting Enzyme Activity and Blood Pressure in Spontaneously Hypertensive Rats.

    PubMed

    Takagaki, Akiko; Nanjo, Fumio

    2015-09-23

    Inhibitory activity of angiotensin I-converting enzyme (ACE) was examined with (-)-epigallocatechin gallate (EGCG) metabolites produced by intestinal bacteria, together with tea catechins. All of the metabolites showed ACE inhibitory activities and the order of IC50 was hydroxyphenyl valeric acids > 5-(3,4,5-trihydroxyphenyl)-γ-valerolactone (1) > trihydroxyphenyl 4-hydroxyvaleric acid ≫ dihydroxyphenyl 4-hydroxyvaleric acid ≫ 5-(3,5-dihydroxyphenyl)-γ-valerolactone (2). Among the catechins, galloylated catechins exhibited stronger ACE inhibitory activity than nongalloylated catechins. Furthermore, the effects of a single oral intake of metabolites 1 and 2 on systolic blood pressure (SBP) were examined with spontaneously hypertensive rats (SHR). Significant decreases in SBP were observed between 2 h after oral administration of 1 (150 mg/kg in SHR) and the control group (p = 0.002) and between 4 h after administration of 2 (200 mg/kg in SHR) and the control group (p = 0.044). These results suggest that the two metabolites have hypotensive effects in vivo.

  19. Normal pancreatic and intestinal enzymes in hypophagic growth-retarded rats that received dorsomedial hypothalamic lesions shortly after weaning.

    PubMed

    Bernardis, L L; Lee, P C; Brooks, S; Lebenthal, E

    1984-08-01

    Male weanling Sprague-Dawley rats received bilateral electrolytic lesions in the dorsomedial hypothalamic nuclei (DMNL rats). Sham-operated rats served as controls. After being fed lab chow for two postoperative weeks, the animals were divided into four groups. One group of DMNL rats and controls received a high-caloric diet (high-fat diet, chocolate chip cookies, 32% sucrose solution, potato chips and marshmallows), whereas another group of DMNL rats and controls continued to receive lab chow. The experiment was terminated on the 185th postoperative day. In accordance with previous findings, DMNL rats, irrespective of diet, were lighter and shorter than controls. In addition, DMNL rats fed junk food were lighter than DMNL rats fed lab chow, and junk-fed controls weighed as much as chow-fed controls. Both DMNL rats and controls fed junk food were also shorter and showed higher carcass fat than their chow-fed counterparts. Also, DMNL rats fed junk food had less carcass fat than junk-fed sham-operated controls, whereas in accordance with previous findings, there was no difference between chow-fed DMNL rats and chow-fed sham-operated controls. Irrespective of diet, DMNL rats ate less calories than their respective sham-operated controls. Both absolute and percent pancreas weight and protein/pancreas were unaffected in DMNL rats but were reduced in both junk-fed groups in comparison with their chow-fed counterparts. Both concentrations and contents of pancreatic trypsinogen, amylase and lipase were unaffected in DMNL rats but total activities of all three enzymes were dramatically reduced in the junk-fed compared with the chow-fed DMNL rats.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Slow onset inhibition of bacterial beta-ketoacyl-acyl carrier protein synthases by thiolactomycin.

    PubMed

    Machutta, Carl A; Bommineni, Gopal R; Luckner, Sylvia R; Kapilashrami, Kanishk; Ruzsicska, Bela; Simmerling, Carlos; Kisker, Caroline; Tonge, Peter J

    2010-02-26

    Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the beta-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices alpha5 and alpha6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100-200 microg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton.

  1. Slow onset inhibition of bacterial beta-ketoacyl-acyl carrier protein synthases by thiolactomycin.

    PubMed

    Machutta, Carl A; Bommineni, Gopal R; Luckner, Sylvia R; Kapilashrami, Kanishk; Ruzsicska, Bela; Simmerling, Carlos; Kisker, Caroline; Tonge, Peter J

    2010-02-26

    Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the beta-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices alpha5 and alpha6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100-200 microg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton. PMID:20018879

  2. 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 within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes. PMID:26899032

  3. Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

    PubMed Central

    Jones, A; Davies, H M; Voelker, T A

    1995-01-01

    Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase. PMID:7734968

  4. Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

    PubMed

    Jones, A; Davies, H M; Voelker, T A

    1995-03-01

    Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase.

  5. 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.

  6. 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. PMID:21071236

  7. Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors

    PubMed Central

    Guasch, Laura; Nicklaus, Marc C.; Meier, Jordan L.

    2015-01-01

    SUMMARY The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the metabolic regulation of lysine acetyltransferase (KAT) enzymes. Using a novel chemoproteomic probe, we identify a previously unreported interaction between fatty acyl-CoAs and KAT enzymes. Further analysis reveals that palmitoyl-CoA is a potent inhibitor of KAT activity and that fatty acyl-CoA precursors reduce cellular acetylation levels. These studies implicate fatty acyl-CoAs as endogenous regulators of histone acetylation, and suggest novel strategies for the investigation and metabolic modulation of epigenetic signaling. PMID:26190825

  8. Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

    PubMed Central

    Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

    1995-01-01

    Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase. PMID:12228351

  9. Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

    PubMed

    Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

    1995-01-01

    Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.

  10. Dietary phenylalanine-improved intestinal barrier health in young grass carp (Ctenopharyngodon idella) is associated with increased immune status and regulated gene expression of cytokines, tight junction proteins, antioxidant enzymes and related signalling molecules.

    PubMed

    Feng, Lin; Li, Wen; Liu, Yang; Jiang, Wei-Dan; Kuang, Sheng-Yao; Jiang, Jun; Tang, Ling; Wu, Pei; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

    2015-08-01

    The present work evaluated the effects of dietary phenylalanine (Phe) on the intestinal immune response, tight junction proteins transcript abundance, and the gene expression of immune- and antioxidant-related signalling molecules in the intestine. In addition, the dietary Phe (and Phe + Tyr) requirement of young grass carp (Ctenopharyngodon idella) was also estimated. Fish were fed fish meal-casein-gelatin based diets (302.3 g crude protein kg(-1)) containing 3.4 (basal diet), 6.1, 9.1, 11.5, 14.0 and 16.8 g Phe kg(-1) with a fixed amount of 10.7 g tyrosine kg(-1) for 8 weeks. The results showed that Phe deficiency or excess Phe reduced the lysozyme and acid phosphatase activities and complement C 3 content in the intestine (P < 0.05). Moreover, zonula occludens-1 (ZO-1), occludin and claudin c mRNA levels were highest in the fish fed the diet containing 11.5 g Phe kg(-1) (P < 0.05). However, claudin 12 and claudin b mRNA levels were not significantly affected by dietary Phe (P > 0.05). Gene expression of interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1), target of rapamycin (TOR) and inhibitor of nuclear factor κBα (IκBα) in proximal intestine (PI), mid intestine (MI) and distal intestine (DI) increased as dietary Phe increased up to 6.1, 9.1, 11.5 and 14.0 g kg(-1), respectively (P < 0.05). However, interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α) and nuclear factor-κB p65 (NF-κB p65) mRNA levels showed opposite tendencies. In addition, the mRNA level of superoxide dismutase (SOD) was significantly lower in the intestinal tissue of the group fed a diet with Phe levels of 16.8 g kg(-1) than in those of other groups (P < 0.05). The expression of NF-E2-related factor 2 (Nrf2) gene was increased as dietary Phe increased up to 9.1 g kg(-1) (P < 0.05). In conclusion, Phe improved intestinal immune status, and regulated gene expression of cytokines, tight junction proteins, antioxidant enzymes, NF-κB p65, IκBα, TOR, and Nrf2 in the fish

  11. Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

    PubMed Central

    Guy, Jodie E.; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

    2011-01-01

    Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

  12. Remote control of regioselectivity in acyl-acyl carrier protein-desaturases.

    PubMed

    Guy, Jodie E; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

    2011-10-01

    Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

  13. Measurement of tissue acyl-CoAs using flow-injection tandem mass spectrometry: acyl-CoA profiles in short-chain fatty acid oxidation defects

    PubMed Central

    Palladino, Andrew A.; Chen, Jie; Kallish, Staci; Stanley, Charles A.; Bennett, Michael J.

    2013-01-01

    The primary accumulating metabolites in fatty acid oxidation defects are intramitochondrial acyl-CoAs. Typically, secondary metabolites such as acylcarnitines, acylglycines and dicarboxylic acids are measured to study these disorders. Methods have not been adapted for tissue acyl-CoA measurement in defects with primarily acyl-CoA accumulation. Our objective was to develop a method to measure fatty acyl-CoA species that are present in tissues of mice with fatty acid oxidation defects using flow-injection tandem mass spectrometry. Following the addition of internal standards of [13C2] acetyl-CoA, [13C8] octanoyl-CoA, and [C17] heptadecanoic CoA, acyl-CoA’s are extracted from tissue samples and are injected directly into the mass spectrometer. Data is acquired using a 506.9 neutral loss scan and multiple reaction-monitoring (MRM). This method can identify all long, medium and short-chain acyl-CoA species in wild type mouse liver including predicted 3-hydroxyacyl-CoA species. We validated the method using liver of the short-chain-acyl-CoA dehydrogenase (SCAD) knock-out mice. As expected, there is a significant increase in [C4] butyryl-CoA species in the SCAD −/− mouse liver compared to wild type. We then tested the assay in liver from the short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) deficient mice to determine the profile of acyl-CoA accumulation in this less predictable model. There was more modest accumulation of medium chain species including 3-hydroxyacyl-CoA’s consistent with the known chain-length specificity of the SCHAD enzyme. PMID:23117082

  14. Efficient mono-acylation of fructose by lipase-catalyzed esterification in ionic liquid co-solvents.

    PubMed

    Li, Lu; Ji, Fangling; Wang, Jingyun; Jiang, Bo; Li, Yachen; Bao, Yongming

    2015-10-30

    Fructose monoesters are eco-friendly nonionic surfactants in various applications. Selective preparation of mono-acylated fructose is challenging due to the multiple hydroxyl sites available for acylation both chemically and enzymatically. Ionic liquids (ILs) have profound impacts not only on the reaction media but also on the catalytic properties of enzymes in the acylation process. In this study, utilizing an IL co-solvent system, selective synthesis of mono-acylated fructose with lauric acid catalyzed by immobilized Candida antarctica lipase B (CALB) was investigated. The imidazolium-based ILs selected as co-solvents with 2-methyl-2-butanol (2M2B) markedly improved the ratios of monolauroyl fructose in the presence of 60% [BMIM][TfO] (v/v) and 20% [BMIM][BF4] (v/v), in which the mono-acylated fructose was 85% and 78% respectively. Based on a Ping-Pong Bi-Bi model, a kinetic equation was fitted, by which the kinetic parameters revealed that the affinity between fructose and acyl-enzyme intermediate was enhanced. The inhibition effect of fructose on free enzyme was weakened in the presence of IL co-solvents. The conformation of CALB binding substrates also changed in the co-solvent system as demonstrated by Fourier transform infrared spectra. These results demonstrated that the variation of CALB kinetic characteristics was a crucial factor for the selectivity of mono-acylation in ILs/2M2B co-solvents.

  15. Efficient mono-acylation of fructose by lipase-catalyzed esterification in ionic liquid co-solvents.

    PubMed

    Li, Lu; Ji, Fangling; Wang, Jingyun; Jiang, Bo; Li, Yachen; Bao, Yongming

    2015-10-30

    Fructose monoesters are eco-friendly nonionic surfactants in various applications. Selective preparation of mono-acylated fructose is challenging due to the multiple hydroxyl sites available for acylation both chemically and enzymatically. Ionic liquids (ILs) have profound impacts not only on the reaction media but also on the catalytic properties of enzymes in the acylation process. In this study, utilizing an IL co-solvent system, selective synthesis of mono-acylated fructose with lauric acid catalyzed by immobilized Candida antarctica lipase B (CALB) was investigated. The imidazolium-based ILs selected as co-solvents with 2-methyl-2-butanol (2M2B) markedly improved the ratios of monolauroyl fructose in the presence of 60% [BMIM][TfO] (v/v) and 20% [BMIM][BF4] (v/v), in which the mono-acylated fructose was 85% and 78% respectively. Based on a Ping-Pong Bi-Bi model, a kinetic equation was fitted, by which the kinetic parameters revealed that the affinity between fructose and acyl-enzyme intermediate was enhanced. The inhibition effect of fructose on free enzyme was weakened in the presence of IL co-solvents. The conformation of CALB binding substrates also changed in the co-solvent system as demonstrated by Fourier transform infrared spectra. These results demonstrated that the variation of CALB kinetic characteristics was a crucial factor for the selectivity of mono-acylation in ILs/2M2B co-solvents. PMID:26343327

  16. Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities.

    PubMed

    Edlin, D A; Kille, P; Wilkinson, M D; Jones, H D; Harwood, J L

    2000-12-01

    We have transformed varieties of wheat with a Pisum sativum glycerol-3-phosphate acyltransferase gene, and also with an Arabidopsis thaliana acyl-ACP thioesterase gene. Morphological (growth, organelle development) and metabolic changes (fatty acid labelling of chloroplast and non-chloroplast lipids) have been observed in transgenics with altered gene expression for either enzyme. PMID:11171169

  17. Modified acyl-ACP desaturase

    DOEpatents

    Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter

    1998-01-06

    Disclosed is a methods 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.

  18. Modified Acyl-ACP desaturase

    DOEpatents

    Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter

    1999-03-30

    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.

  19. Intestinal leiomyoma

    MedlinePlus

    Leiomyoma - intestine ... McLaughlin P, Maher MM. The duodenum and small intestine. In: Adam A, Dixon AK, Gillard JH, Schaefer- ... Roline CE, Reardon RF. Disorders of the small intestine. In: Marx JA, Hockberger RS, Walls RM, et ...

  20. Intestinal Cancer

    MedlinePlus

    ... connects your stomach to your large intestine. Intestinal cancer is rare, but eating a high-fat diet ... increase your risk. Possible signs of small intestine cancer include Abdominal pain Weight loss for no reason ...

  1. Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ...

  2. Deterioration of white croaker (Pennahia argentata) meat thermally-induced gel products caused by proteolytic enzymes in the contaminated intestine and kidney.

    PubMed

    Ueki, Nobuhiko; Wan, Jianrong; Watabe, Shugo

    2016-05-15

    Thermally-induced gels were made from white croaker (Pennahia argentata) meat in the presence of its organ extracts by pre-heating at 40 and 65°C for 20 min and subsequent heating at 85°C for 20 min. The breaking strength of the gels decreased with increasing concentrations of the intestinal extracts accompanying decomposition of myosin heavy chains. However, no significant changes in the gel strength occurred when the kidney extract was added. The proteolytic activity in the intestinal extracts examined in the meat homogenate had a maximum at 60°C and pH 8.90. These results suggest that the intestinal rather than kidney proteolytic activities are responsible for gel softening known as a modori phenomenon. Thus, the removal of intestinal tracts is essential to maintain a high quality of surimi-based products.

  3. Large-scale multiplex absolute protein quantification of drug-metabolizing enzymes and transporters in human intestine, liver, and kidney microsomes by SWATH-MS: Comparison with MRM/SRM and HR-MRM/PRM.

    PubMed

    Nakamura, Kenji; Hirayama-Kurogi, Mio; Ito, Shingo; Kuno, Takuya; Yoneyama, Toshihiro; Obuchi, Wataru; Terasaki, Tetsuya; Ohtsuki, Sumio

    2016-08-01

    The purpose of the present study was to examine simultaneously the absolute protein amounts of 152 membrane and membrane-associated proteins, including 30 metabolizing enzymes and 107 transporters, in pooled microsomal fractions of human liver, kidney, and intestine by means of SWATH-MS with stable isotope-labeled internal standard peptides, and to compare the results with those obtained by MRM/SRM and high resolution (HR)-MRM/PRM. The protein expression levels of 27 metabolizing enzymes, 54 transporters, and six other membrane proteins were quantitated by SWATH-MS; other targets were below the lower limits of quantitation. Most of the values determined by SWATH-MS differed by less than 50% from those obtained by MRM/SRM or HR-MRM/PRM. Various metabolizing enzymes were expressed in liver microsomes more abundantly than in other microsomes. Ten, 13, and eight transporters listed as important for drugs by International Transporter Consortium were quantified in liver, kidney, and intestinal microsomes, respectively. Our results indicate that SWATH-MS enables large-scale multiplex absolute protein quantification while retaining similar quantitative capability to MRM/SRM or HR-MRM/PRM. SWATH-MS is expected to be useful methodology in the context of drug development for elucidating the molecular mechanisms of drug absorption, metabolism, and excretion in the human body based on protein profile information.

  4. Ligand binding to the ACBD6 protein regulates the acyl-CoA transferase reactions in membranes[S

    PubMed Central

    Soupene, Eric; Kuypers, Frans A.

    2015-01-01

    The binding determinants of the human acyl-CoA binding domain-containing protein (ACBD) 6 and its function in lipid renewal of membranes were investigated. ACBD6 binds acyl-CoAs of a chain length of 6 to 20 carbons. The stoichiometry of the association could not be fitted to a 1-to-1 model. Saturation of ACBD6 by C16:0-CoA required higher concentration than less abundant acyl-CoAs. In contrast to ACBD1 and ACBD3, ligand binding did not result in the dimerization of ACBD6. The presence of fatty acids affected the binding of C18:1-CoA to ACBD6, dependent on the length, the degree of unsaturation, and the stereoisomeric conformation of their aliphatic chain. ACBD1 and ACBD6 negatively affected the formation of phosphatidylcholine (PC) and phosphatidylethanolamine in the red blood cell membrane. The acylation rate of lysophosphatidylcholine into PC catalyzed by the red cell lysophosphatidylcholine-acyltransferase 1 protein was limited by the transfer of the acyl-CoA substrate from ACBD6 to the acyltransferase enzyme. These findings provide evidence that the binding properties of ACBD6 are adapted to prevent its constant saturation by the very abundant C16:0-CoA and protect membrane systems from the detergent nature of free acyl-CoAs by controlling their release to acyl-CoA-utilizing enzymes. PMID:26290611

  5. Retrobiosynthetic Approach Delineates the Biosynthetic Pathway and the Structure of the Acyl Chain of Mycobacterial Glycopeptidolipids*

    PubMed Central

    Vats, Archana; Singh, Anil Kumar; Mukherjee, Raju; Chopra, Tarun; Ravindran, Madhu Sudhan; Mohanty, Debasisa; Chatterji, Dipankar; Reyrat, Jean-Marc; Gokhale, Rajesh S.

    2012-01-01

    Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C26-C34 fatty acyl chain that is linked to a tetrapeptide (Phe-Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl-AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs. PMID:22798073

  6. Topology and acylation of spiralin.

    PubMed Central

    Wróblewski, H; Nyström, S; Blanchard, A; Wieslander, A

    1989-01-01

    Of the 51 polypeptides detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the plasma membrane of the helical mollicute Spiroplasma melliferum, 21 are acylated, predominantly with myristic (14:0) and palmitic (16:0) chains. This is notably the case for spiralin, the major membrane protein of this bacterium, which contains an average of 0.7 acyl chains per polypeptide, attached very probably by ester bonds to alcohol amino acids. The amphiphilicity of spiralin was demonstrated by the behavior of the protein in charge-shift electrophoresis, its incorporation into liposomes, and its ability to form in the absence of lipids and detergents, globular protein micelles (diameter, approximately 15 nm). The presence of epitopes on the two faces of the cell membrane, as probed by antibody adsorption and crossed immunoelectrophoresis, and the strong interaction between spiralin and the intracytoplasmic fibrils show that spiralin is a transmembrane protein. The mean hydropathy of the amino acid composition of spiralin (-0.30) is on the hydrophilic side of the scale. Surprisingly, the water-insoluble core of spiralin micelles, which is the putative membrane anchor, has a still more hydrophilic amino acid composition (mean hydropathy, -0.70) and is enriched in glycine and serine residues. Taking into account all these properties, we propose a topological model for spiralin featuring a transbilayer localization with hydrophilic domains protruding on the two faces of the membrane and connected by a small domain embedded within the apolar region of the lipid bilayer. In this model, the membrane anchoring of the protein is strengthened by a covalently bound acyl chain. Images PMID:2768198

  7. Modified Acyl-ACP desaturase

    DOEpatents

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

    1999-03-30

    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. 2 figs.

  8. 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.

  9. Metabolism of acyl-lipids in Chlamydomonas reinhardtii.

    PubMed

    Li-Beisson, Yonghua; Beisson, Fred; Riekhof, Wayne

    2015-05-01

    Microalgae are emerging platforms for production of a suite of compounds targeting several markets, including food, nutraceuticals, green chemicals, and biofuels. Many of these products, such as biodiesel or polyunsaturated fatty acids (PUFAs), derive from lipid metabolism. A general picture of lipid metabolism in microalgae has been deduced from well characterized pathways of fungi and land plants, but recent advances in molecular and genetic analyses of microalgae have uncovered unique features, pointing out the necessity to study lipid metabolism in microalgae themselves. In the past 10 years, in addition to its traditional role as a model for photosynthetic and flagellar motility processes, Chlamydomonas reinhardtii has emerged as a model organism to study lipid metabolism in green microalgae. Here, after summarizing data on total fatty acid composition, distribution of acyl-lipid classes, and major acyl-lipid molecular species found in C. reinhardtii, we review the current knowledge on the known or putative steps for fatty acid synthesis, glycerolipid desaturation and assembly, membrane lipid turnover, and oil remobilization. A list of characterized or putative enzymes for the major steps of acyl-lipid metabolism in C. reinhardtii is included, and subcellular localizations and phenotypes of associated mutants are discussed. Biogenesis and composition of Chlamydomonas lipid droplets and the potential importance of lipolytic processes in increasing cellular oil content are also highlighted.

  10. LuxR homolog-independent gene regulation by acyl-homoserine lactones in Pseudomonas aeruginosa.

    PubMed

    Chugani, Sudha; Greenberg, Everett Peter

    2010-06-01

    Pseudomonas aeruginosa quorum control of gene expression involves three LuxR-type signal receptors LasR, RhlR, and QscR that respond to the LasI- and RhlI-generated acyl-homoserine lactone (acyl-HSL) signals 3OC12-HSL and C4-HSL. We found that a LasR-RhlR-QscR triple mutant responds to acyl-HSLs by regulating at least 37 genes. LuxR homolog-independent activation of the representative genes antA and catB also occurs in the wild type. Expression of antA was influenced the most by C10-HSL and to a lesser extent by other acyl-HSLs, including the P. aeruginosa 3OC12-HSL and C4-HSL signals. The ant and cat operons encode enzymes for the degradation of anthranilate to tricarboxylic acid cycle intermediates. Our results indicate that LuxR homolog-independent acyl-HSL control of the ant and cat operons occurs via regulation of antR, which codes for the transcriptional activator of the ant operon. Although P. aeruginosa has multiple pathways for anthranilate synthesis, one pathway-the kynurenine pathway for tryptophan degradation-is required for acyl-HSL activation of the ant operon. The kynurenine pathway is also the critical source of anthranilate for energy metabolism via the antABC gene products, as well as the source of anthranilate for synthesis of the P. aeruginosa quinolone signal. Our discovery of LuxR homolog-independent responses to acyl-HSLs provides insight into acyl-HSL signaling. PMID:20498077

  11. Novel deletion in a patient with an isolated peroxisoml acyl-CoA oxidase deficiency

    SciTech Connect

    Poll-The, B.T.; Fournier, B.; Clevers, H.; Wanders, R.J.A.

    1994-09-01

    Disorders with defective peroxisome assembly are associated with multiple peroxisomal enzymatic abnormalities. Besides these diseases patients have been described suspected of having a single enzyme defect in the peroxisomal {beta}-oxidation pathway. Laboratory findings for these patients include elevated plasma very long chain fatty acids (VLCFA) and impaired VLCFA oxidation in fibroblasts. Complementation analysis between these patients and those with a proven single enzyme deficiency, using peroxisomal {beta}-oxidation of VLCFA as the criterion for complementation, has been used to show whether the patients are deficient in acyl-CoA oxidase, peroxisomal trifunctional protein or thiolase activity. Fibroblasts from a patient showing the clinical and biochemical abnormalities of isolated acyl-CoA oxidase deficiency (using cell complementation) were analyzed at the molecular level. Isolation of RNA from patient`s fibroblasts was followed by random reverse transcription of RNA and PCR amplification. PCR products were blotted and hybridized with the human acyl-CoA oxidase cDNA. A fragment 150 bp shorter than normal was found. Upon sequencing, exon 7 was found to be deleted leading to a frameshift in the acyl-CoA oxidase mRNA. Southern blot analysis of the patient`s DNA did not reveal any deletion in contrast to two siblings previously reported as having a deletion of at least 17 kb in the acyl-CoA oxidase gene.

  12. 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

    2013-02-01

    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.

  13. Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil

    SciTech Connect

    Triggiani, M.; D'Souza, D.M.; Chilton, F.H. )

    1991-04-15

    The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC.

  14. Mutation of the Enterohemorrhagic Escherichia coli Core LPS Biosynthesis Enzyme RfaD Confers Hypersusceptibility to Host Intestinal Innate Immunity In vivo.

    PubMed

    Kuo, Cheng-Ju; Chen, Jenn-Wei; Chiu, Hao-Chieh; Teng, Ching-Hao; Hsu, Tai-I; Lu, Pei-Jung; Syu, Wan-Jr; Wang, Sin-Tian; Chou, Ting-Chen; Chen, Chang-Shi

    2016-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen causing severe diseases in humans worldwide. Currently, there is no specific treatment available for EHEC infection and the use of conventional antibiotics is contraindicated. Therefore, identification of potential therapeutic targets and development of effective measures to control and treat EHEC infection are needed. Lipopolysaccharides (LPS) are surface glycolipids found on the outer membrane of gram-negative bacteria, including EHEC, and LPS biosynthesis has long been considered as potential anti-bacterial target. Here, we demonstrated that the EHEC rfaD gene that functions in the biosynthesis of the LPS inner core is required for the intestinal colonization and pathogenesis of EHEC in vivo. Disruption of the EHEC rfaD confers attenuated toxicity in Caenorhabditis elegans and less bacterial colonization in the intestine of C. elegans and mouse. Moreover, rfaD is also involved in the control of susceptibility of EHEC to antimicrobial peptides and host intestinal immunity. It is worth noting that rfaD mutation did not interfere with the growth kinetics when compared to the wild-type EHEC cells. Taken together, we demonstrated that mutations of the EHEC rfaD confer hypersusceptibility to host intestinal innate immunity in vivo, and suggested that targeting the RfaD or the core LPS synthesis pathway may provide alternative therapeutic regimens for EHEC infection. PMID:27570746

  15. Mutation of the Enterohemorrhagic Escherichia coli Core LPS Biosynthesis Enzyme RfaD Confers Hypersusceptibility to Host Intestinal Innate Immunity In vivo

    PubMed Central

    Kuo, Cheng-Ju; Chen, Jenn-Wei; Chiu, Hao-Chieh; Teng, Ching-Hao; Hsu, Tai-I; Lu, Pei-Jung; Syu, Wan-Jr; Wang, Sin-Tian; Chou, Ting-Chen; Chen, Chang-Shi

    2016-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen causing severe diseases in humans worldwide. Currently, there is no specific treatment available for EHEC infection and the use of conventional antibiotics is contraindicated. Therefore, identification of potential therapeutic targets and development of effective measures to control and treat EHEC infection are needed. Lipopolysaccharides (LPS) are surface glycolipids found on the outer membrane of gram-negative bacteria, including EHEC, and LPS biosynthesis has long been considered as potential anti-bacterial target. Here, we demonstrated that the EHEC rfaD gene that functions in the biosynthesis of the LPS inner core is required for the intestinal colonization and pathogenesis of EHEC in vivo. Disruption of the EHEC rfaD confers attenuated toxicity in Caenorhabditis elegans and less bacterial colonization in the intestine of C. elegans and mouse. Moreover, rfaD is also involved in the control of susceptibility of EHEC to antimicrobial peptides and host intestinal immunity. It is worth noting that rfaD mutation did not interfere with the growth kinetics when compared to the wild-type EHEC cells. Taken together, we demonstrated that mutations of the EHEC rfaD confer hypersusceptibility to host intestinal innate immunity in vivo, and suggested that targeting the RfaD or the core LPS synthesis pathway may provide alternative therapeutic regimens for EHEC infection. PMID:27570746

  16. Layer-by-Layer Assembled Milk Protein Coated Magnetic Nanoparticle Enabled Oral Drug Delivery with High Stability in Stomach and Enzyme-Responsive Release in Small Intestine

    PubMed Central

    Huang, Jing; Shu, Qing; Wang, Liya; Wu, Hui; Wang, Andrew Y.; Mao, Hui

    2014-01-01

    We report a novel drug delivery system composed of layer-by-layer (LBL) milk protein casein (CN) coated iron oxide nanoparticles. Doxorubicin (DOX) and indocyanine green (ICG) were selected as model drug molecules, which were incorporated into the inner polymeric layer, and subsequently coated with casein. The resulting casein coated iron oxide nanoparticles (CN-DOX/ICG-IO) were stable in the acidic gastric condition with the presence of gastric protease. On the other hand, the loaded drugs were released when the casein outer layer was gradually degraded by the intestinal protease in the simulated intestine condition. Such unique properties enable maintenance of the bioactivity of the drugs and thus enhance the drug delivery efficiency. Ex vivo experiments showed that the LBL CN-DOX-IO improved the translocation of DOX across microvilli and its absorption in the small intestine sacs. In vivo imaging of mice that were orally administered with these LBL CN-ICG-IO nanostructures further confirmed that the reported drug delivery vehicles could pass the stomach without significant degradation, and then accumulated in the small intestine. In addition, the magnetic iron oxide nanoparticle core offered an MRI contrast enhancing capability for in vivo imaging guided drug delivery. Therefore, the reported LBL CN-DOX/ICG-IO is a promising oral drug delivery nanoplatform, especially for drugs that are poorly soluble in water or degradable in the gastric environment. PMID:25477177

  17. Stability-increasing effects of anthocyanin glycosyl acylation.

    PubMed

    Zhao, Chang-Ling; Yu, Yu-Qi; Chen, Zhong-Jian; Wen, Guo-Song; Wei, Fu-Gang; Zheng, Quan; Wang, Chong-De; Xiao, Xing-Lei

    2017-01-01

    This review comprehensively summarizes the existing knowledge regarding the chemical implications of anthocyanin glycosyl acylation, the effects of acylation on the stability of acylated anthocyanins and the corresponding mechanisms. Anthocyanin glycosyl acylation commonly refers to the phenomenon in which the hydroxyl groups of anthocyanin glycosyls are esterified by aliphatic or aromatic acids, which is synthetically represented by the acylation sites as well as the types and numbers of acyl groups. Generally, glycosyl acylation increases the in vitro and in vivo chemical stability of acylated anthocyanins, and the mechanisms primarily involve physicochemical, stereochemical, photochemical, biochemical or environmental aspects under specific conditions. Additionally, the acylation sites as well as the types and numbers of acyl groups influence the stability of acylated anthocyanins to different degrees. This review could provide insight into the optimization of the stability of anthocyanins as well as the application of suitable anthocyanins in food, pharmaceutical and cosmetic industries. PMID:27507456

  18. Intestinal β-galactosidases

    PubMed Central

    Gray, Gary M.; Santiago, Nilda A.; Colver, Eugene H.; Genel, Myron

    1969-01-01

    Despite the high prevalence of intestinal lactase deficiency in some racial groups and in patients with intestinal disease, the biochemical defect has not been characterized. In the preceding paper normal intestine was found to have two lactases with distinctly different pH optima. Therefore, pH activity curves of homogenates from lactase-deficient intestine were studied, and the pH optimum was found to be shifted from the normal of 5.8 to 4.8. Density gradient ultracentrifugation of intestinal material from five lactase-deficient patients demonstrated absence of a lactase with pH optimum 6.0 and molecular weight 280,000. A second lactase with pH optimum 4.5 and molecular weights of 156,000 and 660,000 remained at normal levels accounting for the shift in the pH optimum in whole intestinal homogenates. In addition, three of the five patients had absence of a smaller β-galactosidase (molecular weight 80,000) that had specificity only for synthetic substrates. Although not a lactase, this enzyme had a pH optimum identical with the missing lactase, and its activity was inhibited by lactose in a partially competitive manner suggesting that it is capable of binding lactose. It is possible that this enzyme is a precursor or fragment of the missing lactase. The residual lactase activity provided by the lactase with low pH optimum represents 20-70% of the activity of the missing enzyme, and yet these patients are not able to digest dietary lactose. Thus it appears that the residual enzyme plays no significant role in the hydrolysis of ingested lactose. PMID:5774110

  19. Acyl-acyl-carrier protein: lysomonogalactosyldiacylglycerol acyltransferase from the cyanobacterium Anabaena variabilis.

    PubMed

    Chen, H H; Wickrema, A; Jaworski, J G

    1988-12-16

    Membranes isolated from the cyanobacterium, Anabaena variabilis, and washed free of soluble endogenous constituents, were capable of catalyzing the direct transfer of the acyl group from acyl-acyl-carrier protein to an endogenous lysomonogalactosyldiacylglycerol to form monogalactosyldiacylglycerol. Other glycolipids including monoglucosyldiacylglycerol and digalactosyldiacylglycerol were not products of this reaction. The transfer was not dependent on any added cofactors. Palmitoyl-, stearoyl- and oleoyl-acyl-carrier protein were approximately equally active as substrates. Transfer was exclusively to the C-1 of the glycerol, as demonstrated by hydrolysis of all incorporated acyl groups by the lipase from Rhizopus arrhizus delamar. In addition to the single galactolipid, a second minor reaction product was free fatty acid, presumably due to hydrolysis of the acyl-acyl-carrier protein. Using a double-labelled [14C]acyl-[14C]acyl-carrier protein, the reaction was demonstrated to be a transfer reaction, rather than a simple exchange of acyl groups with endogenous monogalactosyldiacylglycerol. The transfer reaction mechanism was also confirmed by increasing activity with the addition of liposomes of lysomonogalactosyldiacylglycerol.

  20. Novel role of a triglyceride-synthesizing enzyme: DGAT1 at the crossroad between triglyceride and cholesterol metabolism

    PubMed Central

    Sachdev, Vinay; Leopold, Christina; Bauer, Raimund; Patankar, Jay V.; Iqbal, Jahangir; Obrowsky, Sascha; Boverhof, Renze; Doktorova, Marcela; Scheicher, Bernhard; Goeritzer, Madeleine; Kolb, Dagmar; Turnbull, Andrew V.; Zimmer, Andreas; Hoefler, Gerald; Hussain, M. Mahmood; Groen, Albert K.; Kratky, Dagmar

    2016-01-01

    Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol (TG) biosynthesis. Here we show that genetic deficiency and pharmacological inhibition of DGAT1 in mice alters cholesterol metabolism. Cholesterol absorption, as assessed by acute cholesterol uptake, was significantly decreased in the small intestine and liver upon DGAT1 deficiency/inhibition. Ablation of DGAT1 in the intestine (I-DGAT1−/−) alone is sufficient to cause these effects. Consequences of I-DGAT1 deficiency phenocopy findings in whole-body DGAT1−/− and DGAT1 inhibitor-treated mice. We show that deficiency/inhibition of DGAT1 affects cholesterol metabolism via reduced chylomicron size and increased trans-intestinal cholesterol excretion. These effects are independent of cholesterol uptake at the apical surface of enterocytes but mediated through altered dietary fatty acid metabolism. Our findings provide insight into a novel role of DGAT1 and identify a pathway by which intestinal DGAT1 deficiency affects whole-body cholesterol homeostasis in mice. Targeting intestinal DGAT1 may represent a novel approach for treating hypercholesterolemia. PMID:27344248

  1. Novel role of a triglyceride-synthesizing enzyme: DGAT1 at the crossroad between triglyceride and cholesterol metabolism.

    PubMed

    Sachdev, Vinay; Leopold, Christina; Bauer, Raimund; Patankar, Jay V; Iqbal, Jahangir; Obrowsky, Sascha; Boverhof, Renze; Doktorova, Marcela; Scheicher, Bernhard; Goeritzer, Madeleine; Kolb, Dagmar; Turnbull, Andrew V; Zimmer, Andreas; Hoefler, Gerald; Hussain, M Mahmood; Groen, Albert K; Kratky, Dagmar

    2016-09-01

    Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol (TG) biosynthesis. Here we show that genetic deficiency and pharmacological inhibition of DGAT1 in mice alters cholesterol metabolism. Cholesterol absorption, as assessed by acute cholesterol uptake, was significantly decreased in the small intestine and liver upon DGAT1 deficiency/inhibition. Ablation of DGAT1 in the intestine (I-DGAT1(-/-)) alone is sufficient to cause these effects. Consequences of I-DGAT1 deficiency phenocopy findings in whole-body DGAT1(-/-) and DGAT1 inhibitor-treated mice. We show that deficiency/inhibition of DGAT1 affects cholesterol metabolism via reduced chylomicron size and increased trans-intestinal cholesterol excretion. These effects are independent of cholesterol uptake at the apical surface of enterocytes but mediated through altered dietary fatty acid metabolism. Our findings provide insight into a novel role of DGAT1 and identify a pathway by which intestinal DGAT1 deficiency affects whole-body cholesterol homeostasis in mice. Targeting intestinal DGAT1 may represent a novel approach for treating hypercholesterolemia.

  2. Macrocyclic prolinyl acyl guanidines as inhibitors of β-secretase (BACE).

    PubMed

    Boy, Kenneth M; Guernon, Jason M; Wu, Yong-Jin; Zhang, Yunhui; Shi, Joe; Zhai, Weixu; Zhu, Shirong; Gerritz, Samuel W; Toyn, Jeremy H; Meredith, Jere E; Barten, Donna M; Burton, Catherine R; Albright, Charles F; Good, Andrew C; Grace, James E; Lentz, Kimberley A; Olson, Richard E; Macor, John E; Thompson, Lorin A

    2015-11-15

    The synthesis, evaluation, and structure-activity relationships of a class of acyl guanidines which inhibit the BACE-1 enzyme are presented. The prolinyl acyl guanidine chemotype (7c), unlike compounds of the parent isothiazole chemotype (1), yielded compounds with good agreement between their enzymatic and cellular potency as well as a reduced susceptibility to P-gp efflux. Further improvements in potency and P-gp ratio were realized via a macrocyclization strategy. The in vivo profile in wild-type mice and P-gp effects for the macrocyclic analog 21c is presented.

  3. A simple homogeneous scintillation proximity assay for acyl-coenzyme A:diacylglycerol acyltransferase.

    PubMed

    Seethala, Ramakrishna; Peterson, Tara; Dong, Jessica; Chu, Ching-Hsuen; Chen, Luping; Golla, Rajasree; Ma, Zhengping; Panemangalore, Reshma; Lawrence, R Michael; Cheng, Dong

    2008-12-15

    Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is a key enzyme in triacylglycerol synthesis, and inhibiting this enzyme is a promising approach for treating obesity, type II diabetes, and dyslipidemia. There are two distinct DGAT enzymes: DGAT1 and DGAT2. The conventional assay for measuring DGAT activity is a thin layer chromatography (TLC) method, which is not amenable to screening a large number of compounds. To increase the throughput, we have developed a novel, homogeneous scintillation proximity assay (SPA) for DGAT. In this assay, when (3)H-labeled acyl-CoA is used as the acyl donor and diacylglycerol is used as the acyl acceptor, the (3)H-labeled triacylglycerol product formed in the reaction binds to polylysine SPA beads, producing a signal that is measured in a TopCount or LEADseeker. The apparent Michaelis-Menten kinetic parameters determined by this DGAT SPA method agreed well with the values determined with the conventional TLC assay. The statistical values also indicate that the DGAT SPA is a robust assay, with a Z' of more than 0.60 and a signal/background ratio of approximately 9. These results suggest that the current assay provides high-throughput capacity for the identification of DGAT inhibitors.

  4. 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...

  5. Intestinal Malrotation

    MedlinePlus

    ... the intestines don't position themselves normally during fetal development and aren't attached inside properly as a result. The exact reason this occurs is unknown. When a fetus develops in the womb, the intestines start out ...

  6. Intestine Transplant

    MedlinePlus

    ... intestine segment, most intestine transplants involve a whole organ from a deceased donor. In addition, most intestine transplants are performed in ... blood before surgery. I am looking for ... allocation About UNOS Being a living donor Calculator - CPRA Calculator - KDPI Calculator - LAS Calculator - MELD ...

  7. A Liver-Specific Defect of Acyl-CoA Degradation Produces Hyperammonemia, Hypoglycemia and a Distinct Hepatic Acyl-CoA Pattern

    PubMed Central

    Gauthier, Nicolas; Wu, Jiang Wei; Wang, Shu Pei; Allard, Pierre; Mamer, Orval A.; Sweetman, Lawrence; Moser, Ann B.; Kratz, Lisa; Alvarez, Fernando; Robitaille, Yves; Lépine, François; Mitchell, Grant A.

    2013-01-01

    Most conditions detected by expanded newborn screening result from deficiency of one of the enzymes that degrade acyl-coenzyme A (CoA) esters in mitochondria. The role of acyl-CoAs in the pathophysiology of these disorders is poorly understood, in part because CoA esters are intracellular and samples are not generally available from human patients. We created a mouse model of one such condition, deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (HL), in liver (HLLKO mice). HL catalyses a reaction of ketone body synthesis and of leucine degradation. Chronic HL deficiency and acute crises each produced distinct abnormal liver acyl-CoA patterns, which would not be predictable from levels of urine organic acids and plasma acylcarnitines. In HLLKO hepatocytes, ketogenesis was undetectable. Carboxylation of [2-14C] pyruvate diminished following incubation of HLLKO hepatocytes with the leucine metabolite 2-ketoisocaproate (KIC). HLLKO mice also had suppression of the normal hyperglycemic response to a systemic pyruvate load, a measure of gluconeogenesis. Hyperammonemia and hypoglycemia, cardinal features of many inborn errors of acyl-CoA metabolism, occurred spontaneously in some HLLKO mice and were inducible by administering KIC. KIC loading also increased levels of several leucine-related acyl-CoAs and reduced acetyl-CoA levels. Ultrastructurally, hepatocyte mitochondria of KIC-treated HLLKO mice show marked swelling. KIC-induced hyperammonemia improved following administration of carglumate (N-carbamyl-L-glutamic acid), which substitutes for the product of an acetyl-CoA-dependent reaction essential for urea cycle function, demonstrating an acyl-CoA-related mechanism for this complication. PMID:23861731

  8. Protease-catalyzed peptide synthesis using inverse substrates: the influence of reaction conditions on the trypsin acyl transfer efficiency.

    PubMed

    Schellenberger, V; Jakubke, H D; Zapevalova, N P; Mitin, Y V

    1991-06-01

    Benzyloxycarbonyl-L-alanine p-guanidinophenyl ester behaves as a trypsin "inverse substrate," i.e., a cationic center is included in the leaving group instead of being in the acyl moiety. Using this substrate as an acyl donor, trypsin catalyzes the synthesis of peptide bonds that cannot be split by this enzyme. An optimal acyl transfer efficiency was achieved between pH 8 and 9 at 30 degrees C.The addition of as much as 50% cosolvent was shown to be of minor influence on the acyl transfer efficiency, whereas the reaction velocity decreases by more than one order of magnitude. The efficiency of H-Leu-NH(2) and H-Val-NH(2) in deacylation is almost the same for "inverse" and normal type substrates. PMID:18600704

  9. Antifibrotic Activity of Acylated and Unacylated Ghrelin

    PubMed Central

    Angelino, Elia; Reano, Simone; Ferrara, Michele; Agosti, Emanuela; Graziani, Andrea; Filigheddu, Nicoletta

    2015-01-01

    Fibrosis can affect almost all tissues and organs, it often represents the terminal stage of chronic diseases, and it is regarded as a major health issue for which efficient therapies are needed. Tissue injury, by inducing necrosis/apoptosis, triggers inflammatory response that, in turn, promotes fibroblast activation and pathological deposition of extracellular matrix. Acylated and unacylated ghrelin are the main products of the ghrelin gene. The acylated form, through its receptor GHSR-1a, stimulates appetite and growth hormone (GH) release. Although unacylated ghrelin does not bind or activate GHSR-1a, it shares with the acylated form several biological activities. Ghrelin peptides exhibit anti-inflammatory, antioxidative, and antiapoptotic activities, suggesting that they might represent an efficient approach to prevent or reduce fibrosis. The aim of this review is to summarize the available evidence regarding the effects of acylated and unacylated ghrelin on different pathologies and experimental models in which fibrosis is a predominant characteristic. PMID:25960743

  10. Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B.

    PubMed

    Martinelle, M; Hult, K

    1995-09-01

    The acyl transfer reactions catalysed by Candida antartica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm values of the esterification of octanoic acid with different alcohols was 10-30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid. PMID:7669809

  11. Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B.

    PubMed

    Martinelle, M; Hult, K

    1995-09-01

    The acyl transfer reactions catalysed by Candida antartica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm values of the esterification of octanoic acid with different alcohols was 10-30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid.

  12. Immunochemical quantification of cynomolgus CYP2J2, CYP4A and CYP4F enzymes in liver and small intestine.

    PubMed

    Uehara, Shotaro; Murayama, Norie; Nakanishi, Yasuharu; Nakamura, Chika; Hashizume, Takanori; Zeldin, Darryl C; Yamazaki, Hiroshi; Uno, Yasuhiro

    2015-02-01

    1. An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys, an important animal species for drug metabolism studies. 2. In this study, cynomolgus CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, along with CYP2J2, were immunoquantified using selective antibodies in 28 livers and 35 small intestines, and their content was compared with CYP1A, CYP2A, CYP2B6, CYP2C9/19, CYP2D, CYP2E1, CYP3A4 and CYP3A5, previously quantified. 3. In livers, CYP2J2, CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, varied 1.3- to 4.3-fold, represented 11.2, 14.4, 8.0, 2.7 and 0.3% of total immunoquantified CYP1-4 proteins, respectively. 4. In small intestines, CYP2J2, CYP4F2/3, CYP4F11 and CYP4F12, varied 2.4- to 9.7-fold, represented 6.9, 36.4, 2.4 and 9.3% of total immunoquantified CYP1-4 proteins, respectively, making CYP4F the most abundant P450 subfamily in small intestines. CYP4A11 was under the detection limit in all of the samples analyzed. 5. Significant correlations were found in liver for CYP4A11 with lauric acid 11-/12-hydroxylation and for CYP4F2/3 and CYP4F11 with astemizole hydroxylation. 6. This study revealed the relatively abundant contents of cynomolgus CYP2J2, CYP4A11 and CYP4Fs in liver and/or small intestine, suggesting their potential roles for the metabolism of xenobitotics and endogenous substrates.

  13. Immunochemical quantification of cynomolgus CYP2J2, CYP4A and CYP4F enzymes in liver and small intestine.

    PubMed

    Uehara, Shotaro; Murayama, Norie; Nakanishi, Yasuharu; Nakamura, Chika; Hashizume, Takanori; Zeldin, Darryl C; Yamazaki, Hiroshi; Uno, Yasuhiro

    2015-02-01

    1. An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys, an important animal species for drug metabolism studies. 2. In this study, cynomolgus CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, along with CYP2J2, were immunoquantified using selective antibodies in 28 livers and 35 small intestines, and their content was compared with CYP1A, CYP2A, CYP2B6, CYP2C9/19, CYP2D, CYP2E1, CYP3A4 and CYP3A5, previously quantified. 3. In livers, CYP2J2, CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, varied 1.3- to 4.3-fold, represented 11.2, 14.4, 8.0, 2.7 and 0.3% of total immunoquantified CYP1-4 proteins, respectively. 4. In small intestines, CYP2J2, CYP4F2/3, CYP4F11 and CYP4F12, varied 2.4- to 9.7-fold, represented 6.9, 36.4, 2.4 and 9.3% of total immunoquantified CYP1-4 proteins, respectively, making CYP4F the most abundant P450 subfamily in small intestines. CYP4A11 was under the detection limit in all of the samples analyzed. 5. Significant correlations were found in liver for CYP4A11 with lauric acid 11-/12-hydroxylation and for CYP4F2/3 and CYP4F11 with astemizole hydroxylation. 6. This study revealed the relatively abundant contents of cynomolgus CYP2J2, CYP4A11 and CYP4Fs in liver and/or small intestine, suggesting their potential roles for the metabolism of xenobitotics and endogenous substrates. PMID:25138712

  14. Purification and characterization of a novel pumpkin short-chain acyl-coenzyme A oxidase with structural similarity to acyl-coenzyme A dehydrogenases.

    PubMed

    De Bellis, L; Gonzali, S; Alpi, A; Hayashi, H; Hayashi, M; Nishimura, M

    2000-05-01

    A novel pumpkin (Cucurbita pepo) short-chain acyl-coenzyme A (CoA) oxidase (ACOX) was purified to homogeneity by hydrophobic-interaction, hydroxyapatite, affinity, and anion-exchange chromatography. The purified enzyme is a tetrameric protein, consisting of apparently identical 47-kD subunits. The protein structure of this oxidase differs from other plant and mammalian ACOXs, but is similar to the protein structure of mammalian mitochondrial acyl-CoA dehydrogenase (ACDH) and the recently identified plant mitochondrial ACDH. Subcellular organelle separation by sucrose density gradient centrifugation revealed that the enzyme is localized in glyoxysomes, whereas no immunoreactive bands of similar molecular weight were detected in mitochondrial fractions. The enzyme selectively catalyzes the oxidation of CoA esters of fatty acids with 4 to 10 carbon atoms, and exhibits the highest activity on C-6 fatty acids. Apparently, the enzyme has no activity on CoA esters of branched-chain or dicarboxylic fatty acids. The enzyme is slightly inhibited by high concentrations of substrate and it is not inhibited by Triton X-100 at concentrations up to 0.5% (v/v). The characteristics of this novel ACOX enzyme are discussed in relation to other ACOXs and ACDHs. PMID:10806249

  15. Organ culture of fetal rat intestine. Effects on brush border enzyme activities of the combined administration of dexamethasone and cycloheximide or actinomycin D.

    PubMed

    Simon-Assmann, P; Kédinger, M; Grenier, J F; Haffen, K

    1984-01-01

    Jejunum of 19-day fetal rats was explanted in organ culture for 48 h in the presence of dexamethasone (DX) and cycloheximide (CX) or actinomycin D (Act D). The concentrations of both inhibitors which provided maximal responses without any detrimental alteration of the tissue were determined. During the culture period, CX (0.5 microgram/ml) totally abolished the production of both DX-stimulated enzymes (sucrase, maltase, lactase) and DX-insensitive enzymes (aminopeptidase, alkaline phosphatase). On the contrary, Act D at 2 micrograms/ml exhibited differential levels of inhibition related to the enzyme considered: 100% for sucrase and aminopeptidase, 70% for maltase and 50% for lactase. By contrast, alkaline phosphatase was stimulated 100% by Act D. These data suggest that the mechanism by which DX induces sucrase and stimulates maltase activity takes place at the transcriptional level. They also indicate that the basic maturation of at least maltase and lactase activities depends upon the traduction of a preexisting pool of mRNAs. The superinduced alkaline phosphatase activity obtained with Act D supports the notion that an Act D-sensitive repressor may play a role in the maturation process of this enzyme.

  16. Aberrant protein acylation is a common observation in inborn errors of acyl-CoA metabolism.

    PubMed

    Pougovkina, Olga; Te Brinke, Heleen; Wanders, Ronald J A; Houten, Sander M; de Boer, Vincent C J

    2014-09-01

    Inherited disorders of acyl-CoA metabolism, such as defects in amino acid metabolism and fatty acid oxidation can present with severe clinical symptoms either neonatally or later in life, but the pathophysiological mechanisms are often incompletely understood. We now report the discovery of a novel biochemical mechanism that could contribute to the pathophysiology of these disorders. We identified increased protein lysine butyrylation in short-chain acyl-CoA dehydrogenase (SCAD) deficient mice as a result of the accumulation of butyryl-CoA. Similarly, in SCAD deficient fibroblasts, lysine butyrylation was increased. Furthermore, malonyl-CoA decarboxylase (MCD) deficient patient cells had increased levels of malonylated lysines and propionyl-CoA carboxylase (PCC) deficient patient cells had increased propionylation of lysines. Since lysine acylation can greatly impact protein function, aberrant lysine acylation in inherited disorders associated with acyl-CoA accumulation may well play a role in their disease pathophysiology. PMID:24531926

  17. Microbial Tailoring of Acyl Peptidic Siderophores

    PubMed Central

    2015-01-01

    Marine bacteria produce an abundance of suites of acylated siderophores characterized by a unique, species-dependent headgroup that binds iron(III) and one of a series of fatty acid appendages. Marinobacter sp. DS40M6 produces a suite of seven acylated marinobactins, with fatty acids ranging from saturated and unsaturated C12–C18 fatty acids. In the present study, we report that in the late log phase of growth, the fatty acids are hydrolyzed by an amide hydrolase producing the peptidic marinobactin headgroup. Halomonas aquamarina str. DS40M3, another marine bacterium isolated originally from the same sample of open ocean water as Marinobacter sp. DS40M6, produces the acyl aquachelins, also as a suite composed of a peptidic headgroup distinct from that of the marinobactins. In contrast to the acyl marinobactins, hydrolysis of the suite of acyl aquachelins is not detected, even when H. aquamarina str. DS40M3 is grown into the stationary phase. The Marinobacter cell-free extract containing the acyl amide hydrolase is active toward exogenous acyl-peptidic siderophores (e.g., aquachelin C, loihichelin C, as well as octanoyl homoserine lactone used in quorum sensing). Further, when H. aquamarina str. DS40M3 is cultured together with Marinobacter sp. DS40M6, the fatty acids of both suites of siderophores are hydrolyzed, and the aquachelin headgroup is also produced. The present study demonstrates that coculturing bacteria leads to metabolically tailored metabolites compared to growth in a single pure culture, which is interesting given the importance of siderophore-mediated iron acquisition for bacterial growth and that Marinobacter sp. DS40M6 and H. aquamarina str. DS40M3 were isolated from the same sample of seawater. PMID:24735218

  18. Effects of dietary Astragalus polysaccharides (APS) on growth performance, immunological parameters, digestive enzymes, and intestinal morphology of Nile tilapia (Oreochromis niloticus).

    PubMed

    Zahran, Eman; Risha, Engy; Abdelhamid, Fatma; Mahgoub, Hebata Allah; Ibrahim, Tarek

    2014-05-01

    This work investigated the potential immunomodulatory and growth-promoting effects of Astragalus polysaccharides (APS) in Nile tilapia (Oreochromis niloticus). The dietary supplementation with APS (1500 mg/kg of diet) caused a significant increase in growth parameters (initial and final weight, weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR) and feed intake (FI), when compared to non-supplemented control basal diet. In addition, APS upregulated the phagocytic activity, the respiratory burst activity, plasma lysozyme, the bactericidal activity, superoxide dismutase (SOD), glutathione peroxidase (GPx), and amylase activity. However, it had no effect on serum nitric oxide (NO) or Malondialdehyde (MDA) levels. While APS had no effect of intestinal histology, a slight increase in the villi length was recorded. Collectively, our results indicate that dietary APS supplementation could improve the growth performance and the immune parameters of cultured tilapia fish.

  19. Remodeling of host phosphatidylcholine by Chlamydia acyltransferase is regulated by acyl-CoA binding protein ACBD6 associated with lipid droplets

    PubMed Central

    Soupene, Eric; Wang, Derek; Kuypers, Frans A

    2015-01-01

    The bacterial human pathogen Chlamydia trachomatis invades cells as an infectious elementary body (EB). The EB is internalized into a vacuole that is hidden from the host defense mechanism, and is modified to sustain the development of the replicative reticulate body (RB). Inside this parasitophorous compartment, called the inclusion, the pathogen survives supported by an active exchange of nutrients and proteins with the host cell. We show that host lipids are scavenged and modified into bacterial-specific lipids by the action of a shared human-bacterial acylation mechanism. The bacterial acylating enzymes for the essential lipids 1-acyl-sn-glycerol 3-phosphate and 1-acyl-sn-phosphatidylcholine were identified as CT453 and CT775, respectively. Bacterial CT775 was found to be associated with lipid droplets (LDs). During the development of C. trachomatis, the human acyl-CoA carrier hACBD6 was recruited to cytosolic LDs and translocated into the inclusion. hACBD6 protein modulated the activity of CT775 in an acyl-CoA dependent fashion and sustained the activity of the bacterial acyltransferase by buffering the concentration of acyl-CoAs. We propose that disruption of the binding activity of the acyl-CoA carrier might represent a new drug-target to prevent growth of C. trachomatis. PMID:25604091

  20. Trapping the dynamic acyl carrier protein in fatty acid biosynthesis

    PubMed Central

    Nguyen, Chi; Haushalter, Robert W.; Lee, D. John; Markwick, Phineus R. L.; Bruegger, Joel; Caldara-Festin, Grace; Finzel, Kara; Jackson, David R.; Ishikawa, Fumihiro; O’Dowd, Bing; McCammon, J. Andrew; Opella, Stanley J.; Tsai, Shiou-Chuan; Burkart, Michael D.

    2015-01-01

    Acyl carrier protein (ACP) transports the growing fatty acid chain between enzyme domains of fatty acid synthase (FAS) during biosynthesis.1 Because FAS enzymes operate upon ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain.2 The transient nature of ACP-enzyme interactions imposes a major obstacle to gaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to properly study protein-protein interactions. In this work, we describe the application of a mechanism-based probe that allows site-selective covalent crosslinking of AcpP to FabA, the E. coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase. We report the 1.9 Å crystal structure of the crosslinked AcpP=FabA complex as a homo-dimer, in which AcpP exhibits two different conformations likely representing snapshots of ACP in action: the 4′-phosphopantetheine (PPant) group of AcpP first binds an arginine-rich groove of FabA, followed by an AcpP helical conformational change that locks the AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution NMR techniques, including chemical shift perturbations and RDC measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. Combined with molecular dynamics simulations, we show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies will prove general for fatty acid, polyketide and non-ribosomal biosyntheses. Here the foundation is laid for defining the dynamic action of carrier protein activity in primary and secondary metabolism, providing insight into pathways that can play major roles in the treatment of cancer, obesity and infectious

  1. Vitamin A metabolism in the human intestinal Caco-2 cell line

    SciTech Connect

    Quick, T.C.; Ong, D.E. )

    1990-12-01

    The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activites, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with {beta}-carotene, retinyl acetate, or retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte. These data suggest the LRAT may be the physiologically important enzyme for the esterification of retinol in Caco-2 cells.

  2. Intestinal Parasitoses.

    ERIC Educational Resources Information Center

    Lagardere, Bernard; Dumburgier, Elisabeth

    1994-01-01

    Intestinal parasites have become a serious public health problem in tropical countries because of the climate and the difficulty of achieving efficient hygiene. The objectives of this journal issue are to increase awareness of the individual and collective repercussions of intestinal parasites, describe the current conditions of contamination and…

  3. Dietary grape seed tannins: effects of nutritional balance and on some enzymic activities along the crypt-villus axis of rat small intestine.

    PubMed

    Vallet, J; Rouanet, J M; Besançon, P

    1994-01-01

    The aim of the study was to determine the nutritional and intestinal effects of grape seed tannins. For this purpose, tannins were incorporated in diets of rats at levels of 0.2 or 2.0% for 31 days in comparison to a control diet. The animals were pair-fed. Nutritional balances were not affected by feeding 0.2% tannins. At the highest dose (2%) grape seed tannins reduced growth as well as dry matter (DM) and nitrogen (N) digestibility. In rats fed protein-free diets, 2% tannins significantly increased endogenous fecal N. Starch and fat were well digested in all groups of rats. No changes in organ weights were observed. Duodenal alkaline phosphatase activity (AP) was never affected by tannins. On the other hand, in the jejunum, along the vilus-crypt unit, a reduction of AP and sucrase appeared at the tip villus which was balanced by an enhancement of 3H-thymidine incorporation in the middle of the crypt zone, giving evidence of endogenous N loss. This study did not reveal a major toxic effect of tannins except a reduced DM and N digestibility; nevertheless tannins directly interfere with mucosal proteins, thereby stimulating the cell renewal.

  4. Testosterone induction of microsomal acyl-CoA reductase and a cytosolic regulatory protein in mouse preputial glands.

    PubMed

    Lee, T C; Kirk, P; Snyder, F

    1986-01-01

    Alkyl and alk-1-enyl (plasmalogens) ether-linked glycerolipids are prominent components of many mammalian cells; moreover, an acetylated form of an alkyl phospholipid was recently found to possess potent hypotensive, inflammatory and allergic properties. In our studies, preputial glands of mice were selected as a model to investigate the regulation of factors involved in the biosynthesis of ether-linked lipids, since these glands contain high concentrations of ether-linked neutral lipids that are under the influence of hormonal control. We found that a key enzyme in the ether-lipid metabolic pathway, microsomal acyl-CoA reductase that catalyzes the formation of long-chain fatty alcohols (precursor of the O-alkyl chain), was increased 16-fold after injecting testosterone into male, castrated mice. This induction was highly specific, since testosterone did not affect another microsomal enzyme, NADPH-cytochrome c reductase. Based on kinetics of enzyme activity changes, the half-life of acyl-CoA reductase was calculated to be 61-70 h. In addition, the activity of a cytosolic stimulatory protein for the acyl-CoA reductase (but not for a different cytosolic protein, lactate dehydrogenase) was also enhanced in the testosterone-treated, male, castrated mice. These findings indicate that acyl-CoA reductase is an important regulatory enzyme in the reactions that lead to the formation of the ether bond in glycerolipids and that it is modulated through hormonal control. PMID:3940533

  5. Transgenic rice seed expressing flavonoid biosynthetic genes accumulate glycosylated and/or acylated flavonoids in protein bodies

    PubMed Central

    Ogo, Yuko; Mori, Tetsuya; Nakabayashi, Ryo; Saito, Kazuki; Takaiwa, Fumio

    2016-01-01

    Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry was performed. A total of 4392 peaks were detected in both transgenic and non-transgenic rice, 20–40% of which were only detected in transgenic rice. Among these, 82 flavonoids, including 37 flavonols, 11 isoflavones, and 34 flavones, were chemically assigned. Most of the flavonols and isoflavones were O-glycosylated, while many flavones were O-glycosylated and/or C-glycosylated. Several flavonoids were acylated with malonyl, feruloyl, acetyl, and coumaroyl groups. These glycosylated/acylated flavonoids are thought to have been biosynthesized by endogenous rice enzymes using newly synthesized flavonoids whose biosynthesis was catalysed by exogenous enzymes. The subcellular localization of the flavonoids differed depending on the class of aglycone and the glycosylation/acylation pattern. Therefore, flavonoids with the intended aglycones were efficiently produced in rice seeds via the exogenous enzymes introduced, while the flavonoids were variously glycosylated/acylated by endogenous enzymes. The results suggest that rice seeds are useful not only as a production platform for plant-specialized metabolites such as flavonoids but also as a tool for expanding the diversity of flavonoid structures, providing novel, physiologically active substances. PMID:26438413

  6. Isolation of Acyl-CoA:cholesterol acyltransferase inhibitor from Persicaria vulgaris.

    PubMed

    Song, Hye Young; Rho, Mun-Chual; Lee, Seung Woong; Kwon, Oh Eok; Chang, Young-Duck; Lee, Hyun Sun; Kim, Young-Kook

    2002-09-01

    In the course of our search for Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors from natural sources, a new type of ACAT inhibitor was isolated from the methanol extract of Persicaria vulgaris. On the basis of spectral evidence, the structure of the active compound was identified as pheophorbide A. Pheophorbide A inhibited ACAT activity with an IC 50 value of 1.1 microg/ml in an enzyme assay using rat liver microsomes with a dose dependent fashion. PMID:12357403

  7. Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase

    PubMed Central

    Vance, Steven; Tkachenko, Olga; Thomas, Ben; Bassuni, Mona; Hong, Hui; Nietlispach, Daniel; Broadhurst, William

    2016-01-01

    Type I modular polyketide synthases (PKSs) produce polyketide natural products by passing a growing acyl substrate chain between a series of enzyme domains housed within a gigantic multifunctional polypeptide assembly. Throughout each round of chain extension and modification reactions, the substrate stays covalently linked to an acyl carrier protein (ACP) domain. In the present study we report on the solution structure and dynamics of an ACP domain excised from MLSA2, module 9 of the PKS system that constructs the macrolactone ring of the toxin mycolactone, cause of the tropical disease Buruli ulcer. After modification of apo ACP with 4′-phosphopantetheine (Ppant) to create the holo form, 15N nuclear spin relaxation and paramagnetic relaxation enhancement (PRE) experiments suggest that the prosthetic group swings freely. The minimal chemical shift perturbations displayed by Ppant-attached C3 and C4 acyl chains imply that these substrate-mimics remain exposed to solvent at the end of a flexible Ppant arm. By contrast, hexanoyl and octanoyl chains yield much larger chemical shift perturbations, indicating that they interact with the surface of the domain. The solution structure of octanoyl-ACP shows the Ppant arm bending to allow the acyl chain to nestle into a nonpolar pocket, whereas the prosthetic group itself remains largely solvent exposed. Although the highly reduced octanoyl group is not a natural substrate for the ACP from MLSA2, similar presentation modes would permit partner enzyme domains to recognize an acyl group while it is bound to the surface of its carrier protein, allowing simultaneous interactions with both the substrate and the ACP. PMID:26920023

  8. Diacylglycerol Kinase ϵ Is Selective for Both Acyl Chains of Phosphatidic Acid or Diacylglycerol*

    PubMed Central

    Lung, Michael; Shulga, Yulia V.; Ivanova, Pavlina T.; Myers, David S.; Milne, Stephen B.; Brown, H. Alex; Topham, Matthew K.; Epand, Richard M.

    2009-01-01

    The phosphatidylinositol (PI) cycle mediates many cellular events by controlling the metabolism of many lipid second messengers. Diacylglycerol kinase ϵ (DGKϵ) has an important role in this cycle. DGKϵ is the only DGK isoform to show inhibition by its product phosphatidic acid (PA) as well as substrate specificity for sn-2 arachidonoyl-diacylglycerol (DAG). Here, we show that this inhibition and substrate specificity are both determined by selectivity for a combination of the sn-1 and sn-2 acyl chains of PA or DAG, respectively, preferring the most prevalent acyl chain composition of lipids involved specifically in the PI cycle, 1-stearoyl-2-arachidonoyl. Although the difference in rate for closely related lipid species is small, there is a significant enrichment of 1-stearoyl-2-arachidonoyl PI because of the cyclical nature of PI turnover. We also show that the inhibition of DGKϵ by PA is competitive and that the deletion of the hydrophobic segment and cationic cluster of DGKϵ does not affect its selectivity for the acyl chains of PA or DAG. Thus, this active site not only recognizes the lipid headgroup but also a combination of the two acyl chains in PA or DAG. We propose a mechanism of DGKϵ regulation where its dual acyl chain selectivity is used to negatively regulate its enzymatic activity in a manner that ensures DGKϵ remains committed to the PI turnover cycle. This novel mechanism of enzyme regulation within a signaling pathway could serve as a template for the regulation of enzymes in other pathways in the cell. PMID:19744926

  9. 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.

  10. Acylation in trypanosomatids: an essential process and potential drug target

    PubMed Central

    Goldston, Amanda M.; Sharma, Aabha I.; Paul, Kimberly S.; Engman, David M.

    2014-01-01

    Fatty acylation—the addition of fatty acid moieties such as myristate and palmitate to proteins—is essential for the survival, growth, and infectivity of the trypanosomatids: Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. Myristoylation and palmitoylation are critical for parasite growth, targeting and localization, and the intrinsic function of some proteins. The trypanosomatids possess a single N-myristoyltransferase (NMT) and multiple palmitoyl acyltransferases, and these enzymes and their cellular targets are only now being characterized. Global inhibition of either process leads to cell death in trypanosomatids, and genetic ablation of NMT compromises virulence. Moreover, NMT inhibitors effectively cure T. brucei infection in rodents. Thus, protein acylation represents an attractive target for the development of trypanocidal drugs. PMID:24954795

  11. Identification of N-Acyl Phosphatidylserine Molecules in Eukaryotic Cells

    PubMed Central

    Guan, Ziqiang; Li, Shengrong; Smith, Dale C.; Shaw, Walter A.; Raetz, Christian R. H.

    2008-01-01

    While profiling the lipidome of the mouse brain by mass spectrometry, we discovered a novel family of N-acyl phosphatidylserine (N-acyl-PS) molecules. These N-acyl-PS species were enriched by DEAE-cellulose column chromatography, and they were then characterized by accurate mass measurements, tandem mass spectrometry, liquid chromatography/mass spectrometry, and comparison to an authentic standard. Mouse brain N-acyl-PS molecules are heterogeneous and constitute about 0.1 % of the total lipid. In addition to various ester-linked fatty acyl chains on their glycerol backbones, the complexity of the N-acyl-PS series is further increased by the presence of diverse amide-linked N-acyl chains, which include saturated, mono-unsaturated and poly-unsaturated species. N-acyl-PS molecular species were also detected in the lipids of pig brain, mouse RAW264.7 macrophage tumor cells and yeast, but not E. coli. N-acyl-PSs may be biosynthetic precursors of N-acyl serine molecules, such as the recently reported signaling lipid N-arachidonoyl serine from bovine brain. We suggest that a phospholipase D might cleave N-acyl-PS to generate N-acyl serine, in analogy to the biosynthesis of the endocannabinoid N-arachidonoyl ethanolamine (anadamide) from N-arachidonoyl phosphatidylethanolamine. PMID:18031065

  12. Structure of YciA from Haemophilus influenzae (HI0827), a Hexameric Broad Specificity Acyl-Coenzyme A Thioesterase

    SciTech Connect

    Willis, Mark A.; Zhuang, Zhihao; Song, Feng; Howard, Andrew; Dunaway-Mariano, Debra; Herzberg, Osnat

    2008-04-02

    The crystal structure of HI0827 from Haemophilus influenzae Rd KW20, initially annotated 'hypothetical protein' in sequence databases, exhibits an acyl-coenzyme A (acyl-CoA) thioesterase 'hot dog' fold with a trimer of dimers oligomeric association, a novel assembly for this enzyme family. In studies described in the preceding paper [Zhuang, Z., Song, F., Zhao, H., Li, L., Cao, J., Eisenstein, E., Herzberg, O., and Dunaway-Mariano, D. (2008) Biochemistry 47, 2789-2796], HI0827 is shown to be an acyl-CoA thioesterase that acts on a wide range of acyl-CoA compounds. Two substrate binding sites are located across the dimer interface. The binding sites are occupied by two CoA molecules, one with full occupancy and the second only partially occupied. The CoA molecules, acquired from HI0827-expressing Escherichia coli cells, remained tightly bound to the enzyme through the protein purification steps. The difference in CoA occupancies indicates a different substrate affinity for each of the binding sites, which in turn implies that the enzyme might be subject to allosteric regulation. Mutagenesis studies have shown that the replacement of the putative catalytic carboxylate Asp44 with an alanine residue abolishes activity. The impact of this mutation is seen in the crystal structure of D44A HI0827. Whereas the overall fold and assembly of the mutant protein are the same as those of the wild-type enzyme, the CoA ligands are absent. The dimer interface is perturbed, and the channel that accommodates the thioester acyl chain is more open and wider than that observed in the wild-type enzyme. A model of intact substrate bound to wild-type HI0827 provides a structural rationale for the broad substrate range.

  13. Intestinal obstruction

    MedlinePlus

    ... of the bowel may be due to: A mechanical cause, which means something is in the way ... lung disease Use of certain medicines, especially narcotics Mechanical causes of intestinal obstruction may include: Adhesions or ...

  14. Degradation of endogenous bacterial cell wall polymers by the muralytic enzyme mutanolysin prevents hepatobiliary injury in genetically susceptible rats with experimental intestinal bacterial overgrowth.

    PubMed Central

    Lichtman, S N; Okoruwa, E E; Keku, J; Schwab, J H; Sartor, R B

    1992-01-01

    Jejunal self-filling blind loops with subsequent small bowel bacterial overgrowth (SBBO) induce hepatobiliary injury in genetically susceptible Lewis rats. Lesions consist of portal tract inflammation, bile duct proliferation, and destruction. To determine the pathogenesis of SBBO-induced hepatobiliary injury, we treated Lewis rats with SBBO by using several agents with different mechanisms of activity. Buffer treatment, ursodeoxycholic acid, prednisone, methotrexate, and cyclosporin A failed to prevent SBBO-induced injury as demonstrated by increased plasma aspartate aminotransferase (AST) and elevated histology scores. However, hepatic injury was prevented by mutanolysin, a muralytic enzyme whose only known activity is to split the beta 1-4 N-acetylmuramyl-N-acetylglucosamine linkage of peptidoglycan-polysaccharide (PG-PS), a bacterial cell wall polymer with potent inflammatory and immunoregulatory properties. Mutanolysin therapy started on the day blind loops were surgically created and continued for 8 wk significantly diminished AST (101 +/- 37 U/liter) and liver histology scores (2.2 +/- 2.7) compared to buffer-treated rats (228 +/- 146 U/liter, P < 0.05, 8.2 +/- 1.9, P < 0.001 respectively). Mutanolysin treatment started during the early phase of hepatic injury, 16-21 d after surgery, decreased AST in 7 of 11 rats from 142 +/- 80 to 103 +/- 24 U/liter contrasted to increased AST in 9 of 11 buffer-treated rats from 108 +/- 52 to 247 +/- 142 U/liter, P < 0.05. Mutanolysin did not change total bacterial numbers within the loop, eliminate Bacteroides sp., have in vitro antibiotic effects, or diminish mucosal PG-PS transport. However, mutanolysin treatment prevented elevation of plasma anti-PG antibodies and tumor necrosis factor-alpha (TNF alpha) levels which occurred in buffer treated rats with SBBO and decreased TNF alpha production in isolated Kupffer cells stimulated in vitro with PG-PS. Based on the preventive and therapeutic activity of this highly specific

  15. Head-group acylation of monogalactosyldiacylglycerol is a common stress response, and the acyl-galactose acyl composition varies with the plant species and applied stress.

    PubMed

    Vu, Hieu Sy; Roth, Mary R; Tamura, Pamela; Samarakoon, Thilani; Shiva, Sunitha; Honey, Samuel; Lowe, Kaleb; Schmelz, Eric A; Williams, Todd D; Welti, Ruth

    2014-04-01

    Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG (galactose-acylated monogalactosyldiacylglycerol) depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses. PMID:24286212

  16. N-Methyltaurine N-acyl amidated bile acids and deoxycholic acid in the bile of angelfish (Pomacanthidae): a novel bile acid profile in Perciform fish.

    PubMed

    Satoh Née Okihara, Rika; Saito, Tetsuya; Ogata, Hiroaki; Ohsaki, Ayumi; Iida, Takashi; Asahina, Kiyoshi; Mitamura, Kuniko; Ikegawa, Shigeo; Hofmann, Alan F; Hagey, Lee R

    2014-02-01

    Two novel N-acyl amidated bile acids, N-methyltaurine conjugated cholic acid and N-methyltaurine conjugated deoxycholic acid, were found to be major biliary bile acids in two species of angelfish the regal (Pygoplites diacanthus) and the blue-girdled (Pomacanthus navarchus) angelfish. The identification was based on their having MS and NMR spectra identical to those of synthetic standards. A survey of biliary bile acids of 10 additional species of angelfish found 7 with N-methyltaurine conjugation. In all 12 species, conjugated deoxycholic acid (known to be formed by bacterial 7-dehydroxylation of cholic acid) was a major bile acid. In all previous studies of biliary bile acids in fish, deoxycholic acid has been present in only trace proportions. In addition, bile acid conjugation with N-methyltaurine has not been detected previously in any known vertebrate. N-methyltaurine conjugated bile acids are resistant to bacterial deconjugation and dehydroxylation, and such resistance to bacterial enzymes should aid in the maintenance of high concentrations of bile acids during lipid digestion. Our findings suggest that these species of angelfish have a novel microbiome in their intestine containing anaerobic bacteria, and describe the presence of N-methyltaurine conjugated bile acids that are resistant to bacterial attack.

  17. Kinetic and structural basis for acyl-group selectivity and NAD+-dependence in Sirtuin-catalyzed deacylation

    PubMed Central

    Thelen, Julie N.; Ito, Akihiro; Yoshida, Minoru; Denu, John M.

    2015-01-01

    Acylation of lysine is an important protein modification regulating diverse biological processes. It was recently demonstrated that members of the human Sirtuin family are capable of catalyzing long-chain deacylation, in addition to the well-known NAD+-dependent deacetylation activity.1 Here we provide a detailed kinetic and structural analysis that describes the interdependence of NAD+ and acyl-group length for a diverse series of human Sirtuins, SIRT1, SIRT2, SIRT3 and SIRT6. Steady-state and rapid-quench kinetic analyses indicated that differences in NAD+ saturation and susceptibility to nicotinamide inhibition reflect unique kinetic behavior displayed by each Sirtuin and depend on acyl-substrate chain length. Though the rate of nucleophilic attack of the 2′-hydroxyl on the C1′-O-alkylimidate intermediate varies with acyl substrate chain length, this step remains rate-determining for SIRT2 and SIRT3; however for SIRT6, this step is no longer rate-limiting for long-chain substrates. Co-crystallization of SIRT2 with myristoylated peptide and NAD+ yielded a co-complex structure with reaction product 2′-O-myristoyl-ADP-ribose, revealing a latent hydrophobic cavity to accommodate the long chain acyl group, and suggesting a general mechanism for long chain deacylation. Comparing two separately solved co-complex structures containing either a myristoylated peptide or 2′-O-myristoyl-ADP-ribose indicate there are conformational changes at the myristoyl-ribose linkage with minimal structural differences in the enzyme active site. During the deacylation reaction, the fatty acyl group is held in a relatively fixed position. We describe a kinetic and structural model to explain how various Sirtuins display unique acyl-substrate preferences and how different reaction kinetics influence NAD+ dependence. The biological implications are discussed. PMID:25897714

  18. 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

  19. Peroxisomal bifunctional enzyme deficiency.

    PubMed Central

    Watkins, P A; Chen, W W; Harris, C J; Hoefler, G; Hoefler, S; Blake, D C; Balfe, A; Kelley, R I; Moser, A B; Beard, M E

    1989-01-01

    Peroxisomal function was evaluated in a male infant with clinical features of neonatal adrenoleukodystrophy. Very long chain fatty acid levels were elevated in both plasma and fibroblasts, and beta-oxidation of very long chain fatty acids in cultured fibroblasts was significantly impaired. Although the level of the bile acid intermediate trihydroxycoprostanoic acid was slightly elevated in plasma, phytanic acid and L-pipecolic acid levels were normal, as was plasmalogen synthesis in cultured fibroblasts. The latter three parameters distinguish this case from classical neonatal adrenoleukodystrophy. In addition, electron microscopy and catalase subcellular distribution studies revealed that, in contrast to neonatal adrenoleukodystrophy, peroxisomes were present in the patient's tissues. Immunoblot studies of peroxisomal beta-oxidation enzymes revealed that the bifunctional enzyme (enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase) was deficient in postmortem liver samples, whereas acyl-CoA oxidase and the mature form of beta-ketothiolase were present. Density gradient centrifugation of fibroblast homogenates confirmed that intact peroxisomes were present. Immunoblots of fibroblasts peroxisomal fractions showed that they contained acyl-CoA oxidase and beta-ketothiolase, but bifunctional enzyme was not detected. Northern analysis, however, revealed that mRNA coding for the bifunctional enzyme was present in the patient's fibroblasts. These results indicate that the primary biochemical defect in this patient is a deficiency of peroxisomal bifunctional enzyme. It is of interest that the phenotype of this patient resembled neonatal adrenoleukodystrophy and would not have been distinguished from this disorder by clinical study alone. Images PMID:2921319

  20. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  1. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

    2011-02-18

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  2. Acylated but not des-acyl ghrelin is neuroprotective in an MPTP mouse model of Parkinson's disease.

    PubMed

    Bayliss, Jacqueline A; Lemus, Moyra; Santos, Vanessa V; Deo, Minh; Elsworth, John D; Andrews, Zane B

    2016-05-01

    The gut hormone ghrelin is widely beneficial in many disease states. However, ghrelin exists in two distinctive isoforms, each with its own metabolic profile. In Parkinson's Disease (PD) acylated ghrelin administration is neuroprotective, however, the role of des-acylated ghrelin remains unknown. In this study, we wanted to identify the relative contribution each isoform plays using the MPTP model of PD. Chronic administration of acylated ghrelin in mice lacking both isoforms of ghrelin (Ghrelin KO) attenuated the MPTP-induced loss on tyrosine hydroxylase (TH) neuronal number and volume and TH protein expression in the nigrostriatal pathway. Moreover, acylated ghrelin reduced the increase in glial fibrillary acidic protein and Ionized calcium binding adaptor molecule 1 microglia in the substantia nigra. However, injection of acylated ghrelin also elevated plasma des-acylated ghrelin, indicating in vivo deacetylation. Next, we chronically administered des-acylated ghrelin to Ghrelin KO mice and observed no neuroprotective effects in terms of TH cell number, TH protein expression, glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 cell number. The lack of a protective effect was mirrored in ghrelin-O-acyltransferase KO mice, which lack the ability to acylate ghrelin and consequently these mice have chronically increased plasma des-acyl ghrelin. Plasma corticosterone was elevated in ghrelin-O-acyltransferase KO mice and with des-acylated ghrelin administration. Overall, our studies suggest that acylated ghrelin is the isoform responsible for in vivo neuroprotection and that pharmacological approaches preventing plasma conversion from acyl ghrelin to des-acyl ghrelin may have clinical efficacy to help slow or prevent the debilitating effects of PD. Ghrelin exists in the plasma as acyl and des-acyl ghrelin. We determined the form responsible for in vivo neuroprotection in a mouse model of Parkinson's disease. Although exogenous acyl ghrelin

  3. Two Clades of Type-1 Brassica napus Diacylglycerol Acyltransferase Exhibit Differences in Acyl-CoA Preference.

    PubMed

    Greer, Michael S; Pan, Xue; Weselake, Randall J

    2016-06-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to produce triacylglycerol, which is the main component of the seed oil of Brassica oilseed species. Phylogenetic analysis of the amino acid sequences encoded by four transcriptionally active DGAT1 genes from Brassica napus suggests that the gene forms diverged over time into two clades (I and II), with representative members in each genome (A and C). The majority of the amino acid sequence differences in these forms of DGAT1, however, reside outside of motifs suggested to be involved in catalysis. Despite this, the clade II enzymes displayed a significantly enhanced preference for linoleoyl-CoA when assessed using in-vitro enzyme assays with yeast microsomes containing recombinant enzyme forms. These findings contribute to our understanding of triacylglycerol biosynthesis in B. napus, and may advance our ability to engineer DGAT1s with desired substrate selectivity properties. PMID:27138895

  4. Identification and characterization of alkenyl hydrolase (lysoplasmalogenase) in microsomes and identification of a plasmalogen-active phospholipase A2 in cytosol of small intestinal epithelium.

    PubMed

    Jurkowitz, M S; Horrocks, L A; Litsky, M L

    1999-02-25

    A lysoplasmalogenase (EC 3.3.2.2; EC 3.3.2.5) that liberates free aldehyde from 1-alk-1'-enyl-sn-glycero-3-phospho-ethanolamine or -choline (lysoplasmalogen) was identified and characterized in rat gastrointestinal tract epithelial cells. Glycerophosphoethanolamine was produced in the reaction in equimolar amounts with the free aldehyde. The microsomal membrane associated enzyme was present throughout the length of the small intestines, with the highest activity in the jejunum and proximal ileum. The rate of alkenyl ether bond hydrolysis was dependent on the concentrations of microsomal protein and substrate, and was linear with respect to time. The enzyme hydrolyzed both ethanolamine- and choline-lysoplasmalogens with similar affinities; the Km values were 40 and 66 microM, respectively. The enzyme had no activity with 1-alk-1'-enyl-2-acyl-sn-glycero-3-phospho-ethanolamine or -choline (intact plasmalogen), thus indicating enzyme specificity for a free hydroxyl group at the sn-2 position. The specific activities were 70 nmol/min/mg protein and 57 nmol/min/mg protein, respectively, for ethanolamine- and choline-lysoplasmalogen. The pH optimum was between 6.8 and 7.4. The enzyme required no known cofactors and was not affected by low mM levels of Ca2+, Mg2+, EDTA, or EGTA. The detergents, Triton X-100, deoxycholate, and octyl glucoside inhibited the enzyme. The chemical and physical properties of the lysoplasmalogenase were very similar to those of the enzyme in liver and brain microsomes. In developmental studies the specific activities of the small intestinal and liver enzymes increased markedly, 11.1- and 3.4-fold, respectively, in the first approximately 40 days of postnatal life. A plasmalogen-active phospholipase A2 activity was identified in the cytosol of the small intestines (3.3 nmol/min/mg protein) and liver (0.3 nmol/min/mg protein) using a novel coupled enzyme assay with microsomal lysoplasmalogenase as the coupling enzyme.

  5. Acyl Coenzyme A Thioesterase Them5/Acot15 Is Involved in Cardiolipin Remodeling and Fatty Liver Development

    PubMed Central

    Gut, Heinz; Hynx, Debby; Marcellin, David; Bleck, Christopher K. E.; Genoud, Christel; Cron, Peter; Keusch, Jeremy J.; Dummler, Bettina; Esposti, Mauro Degli

    2012-01-01

    Acyl coenzyme A (acyl-CoA) thioesterases hydrolyze thioester bonds in acyl-CoA metabolites. The majority of mammalian thioesterases are α/β-hydrolases and have been studied extensively. A second class of Hotdog-fold enzymes has been less well described. Here, we present a structural and functional analysis of a new mammalian mitochondrial thioesterase, Them5. Them5 and its paralog, Them4, adopt the classical Hotdog-fold structure and form homodimers in crystals. In vitro, Them5 shows strong thioesterase activity with long-chain acyl-CoAs. Loss of Them5 specifically alters the remodeling process of the mitochondrial phospholipid cardiolipin. Them5−/− mice show deregulation of lipid metabolism and the development of fatty liver, exacerbated by a high-fat diet. Consequently, mitochondrial morphology is affected, and functions such as respiration and β-oxidation are impaired. The novel mitochondrial acyl-CoA thioesterase Them5 has a critical and specific role in the cardiolipin remodeling process, connecting it to the development of fatty liver and related conditions. PMID:22586271

  6. Acyl coenzyme A thioesterase Them5/Acot15 is involved in cardiolipin remodeling and fatty liver development.

    PubMed

    Zhuravleva, Elena; Gut, Heinz; Hynx, Debby; Marcellin, David; Bleck, Christopher K E; Genoud, Christel; Cron, Peter; Keusch, Jeremy J; Dummler, Bettina; Esposti, Mauro Degli; Hemmings, Brian A

    2012-07-01

    Acyl coenzyme A (acyl-CoA) thioesterases hydrolyze thioester bonds in acyl-CoA metabolites. The majority of mammalian thioesterases are α/β-hydrolases and have been studied extensively. A second class of Hotdog-fold enzymes has been less well described. Here, we present a structural and functional analysis of a new mammalian mitochondrial thioesterase, Them5. Them5 and its paralog, Them4, adopt the classical Hotdog-fold structure and form homodimers in crystals. In vitro, Them5 shows strong thioesterase activity with long-chain acyl-CoAs. Loss of Them5 specifically alters the remodeling process of the mitochondrial phospholipid cardiolipin. Them5(-/-) mice show deregulation of lipid metabolism and the development of fatty liver, exacerbated by a high-fat diet. Consequently, mitochondrial morphology is affected, and functions such as respiration and β-oxidation are impaired. The novel mitochondrial acyl-CoA thioesterase Them5 has a critical and specific role in the cardiolipin remodeling process, connecting it to the development of fatty liver and related conditions. PMID:22586271

  7. 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

  8. Acyl glucuronides: the good, the bad and the ugly.

    PubMed

    Regan, Sophie L; Maggs, James L; Hammond, Thomas G; Lambert, Craig; Williams, Dominic P; Park, B Kevin

    2010-10-01

    Acyl glucuronidation is the major metabolic conjugation reaction of most carboxylic acid drugs in mammals. The physiological consequences of this biotransformation have been investigated incompletely but include effects on drug metabolism, protein binding, distribution and clearance that impact upon pharmacological and toxicological outcomes. In marked contrast, the exceptional but widely disparate chemical reactivity of acyl glucuronides has attracted far greater attention. Specifically, the complex transacylation and glycation reactions with proteins have provoked much inconclusive debate over the safety of drugs metabolised to acyl glucuronides. It has been hypothesised that these covalent modifications could initiate idiosyncratic adverse drug reactions. However, despite a large body of in vitro data on the reactions of acyl glucuronides with protein, evidence for adduct formation from acyl glucuronides in vivo is limited and potentially ambiguous. The causal connection of protein adduction to adverse drug reactions remains uncertain. This review has assessed the intrinsic reactivity, metabolic stability and pharmacokinetic properties of acyl glucuronides in the context of physiological, pharmacological and toxicological perspectives. Although numerous experiments have characterised the reactions of acyl glucuronides with proteins, these might be attenuated substantially in vivo by rapid clearance of the conjugates. Consequently, to delineate a relationship between acyl glucuronide formation and toxicological phenomena, detailed pharmacokinetic analysis of systemic exposure to the acyl glucuronide should be undertaken adjacent to determining protein adduct concentrations in vivo. Further investigation is required to ascertain whether acyl glucuronide clearance is sufficient to prevent covalent modification of endogenous proteins and consequentially a potential immunological response. PMID:20830700

  9. Efficient regioselective acylation of quercetin using Rhizopus oryzae lipase and its potential as antioxidant.

    PubMed

    Kumar, Vinod; Jahan, Firdaus; Mahajan, Richi V; Saxena, Rajendra Kumar

    2016-10-01

    The present investigation describes the regioselective enzymatic acylation of quercetin with ferulic acid using Rhizopus oryzae lipase. Optimization of reaction parameters resulted in 93.2% yield of the ester synthesized using 750IU of lipase in cyclo-octane at a temperature of 45°C. The reaction was successfully carried out upto 25g scale. The ester synthesized was analyzed by (1)H Nuclear magnetic resonance spectroscopy. The ester synthesized (quercetin ferulate) showed higher antiradical activity as compared to ascorbic acid using the 2,2-diphenyl-1-picrylhydrazyl radical method. These results on enzyme-catalyzed acylation of quercetin might be used to prepare and scale-up other flavonoids derivatives. PMID:27372535

  10. Efficient regioselective acylation of quercetin using Rhizopus oryzae lipase and its potential as antioxidant.

    PubMed

    Kumar, Vinod; Jahan, Firdaus; Mahajan, Richi V; Saxena, Rajendra Kumar

    2016-10-01

    The present investigation describes the regioselective enzymatic acylation of quercetin with ferulic acid using Rhizopus oryzae lipase. Optimization of reaction parameters resulted in 93.2% yield of the ester synthesized using 750IU of lipase in cyclo-octane at a temperature of 45°C. The reaction was successfully carried out upto 25g scale. The ester synthesized was analyzed by (1)H Nuclear magnetic resonance spectroscopy. The ester synthesized (quercetin ferulate) showed higher antiradical activity as compared to ascorbic acid using the 2,2-diphenyl-1-picrylhydrazyl radical method. These results on enzyme-catalyzed acylation of quercetin might be used to prepare and scale-up other flavonoids derivatives.

  11. A thiolate anion buried within the hydrocarbon ruler perturbs PagP lipid acyl chain selection.

    PubMed

    Khan, M Adil; Moktar, Joel; Mott, Patrick J; Bishop, Russell E

    2010-03-23

    The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP exhibits remarkable selectivity because its binding pocket for lipid acyl chains excludes those differing in length from palmitate by a solitary methylene unit. This narrow detergent-binding hydrophobic pocket buried within the eight-strand antiparallel beta-barrel is known as the hydrocarbon ruler. Gly88 lines the acyl chain binding pocket floor, and its substitution can raise the floor to correspondingly shorten the selected acyl chain. An aromatic exciton interaction between Tyr26 and Trp66 provides an intrinsic spectroscopic probe located immediately adjacent to Gly88. The Gly88Cys PagP enzyme was engineered to function as a dedicated myristoyltransferase, but the mutant enzyme instead selected both myristoyl and pentadecanoyl groups, was devoid of the exciton, and displayed a 21 degrees C reduction in thermal stability. We now demonstrate that the structural perturbation results from a buried thiolate anion attributed to suppression of the Cys sulfhydryl group pK(a) from 9.4 in aqueous solvent to 7.5 in the hydrocarbon ruler microenvironment. The Cys thiol is sandwiched at the interface between a nonpolar and a polar beta-barrel interior milieu, suggesting that local electrostatics near the otherwise hydrophobic hydrocarbon ruler pocket serve to perturb the thiol pK(a). Neutralization of the Cys thiolate anion by protonation restores wild-type exciton and thermal stability signatures to Gly88Cys PagP, which then functions as a dedicated myristoyltransferase at pH 7. Gly88Cys PagP assembled in bacterial membranes recapitulates lipid A myristoylation in vivo. Hydrocarbon ruler-exciton coupling in PagP thus reveals a thiol-thiolate ionization mechanism for modulating lipid acyl chain selection.

  12. Head-group acylation of monogalactosyldiacylglycerol is a common stress response, and the acyl-galactose acyl composition varies with the plant species and applied stress

    PubMed Central

    Vu, Hieu Sy; Roth, Mary R.; Tamura, Pamela; Samarakoon, Thilani; Shiva, Sunitha; Honey, Samuel; Lowe, Kaleb; Schmelz, Eric A.; Williams, Todd D.; Welti, Ruth

    2014-01-01

    Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection, and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses. PMID:24286212

  13. Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

    PubMed Central

    Bharathi, Sivakama S.; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E.; Rardin, Matthew J.; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W.; Hirschey, Matthew D.; Goetzman, Eric S.

    2013-01-01

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

  14. Sirtuin 3 (SIRT3) protein regulates long-chain acyl-CoA dehydrogenase by deacetylating conserved lysines near the active site.

    PubMed

    Bharathi, Sivakama S; Zhang, Yuxun; Mohsen, Al-Walid; Uppala, Radha; Balasubramani, Manimalha; Schreiber, Emanuel; Uechi, Guy; Beck, Megan E; Rardin, Matthew J; Vockley, Jerry; Verdin, Eric; Gibson, Bradford W; Hirschey, Matthew D; Goetzman, Eric S

    2013-11-22

    Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases. PMID:24121500

  15. Acquired causes of intestinal malabsorption.

    PubMed

    van der Heide, F

    2016-04-01

    This review focuses on the acquired causes, diagnosis, and treatment of intestinal malabsorption. Intestinal absorption is a complex process that depends on many variables, including the digestion of nutrients within the intestinal lumen, the absorptive surface of the small intestine, the membrane transport systems, and the epithelial absorptive enzymes. Acquired causes of malabsorption are classified by focussing on the three phases of digestion and absorption: 1) luminal/digestive phase, 2) mucosal/absorptive phase, and 3) transport phase. Most acquired diseases affect the luminal/digestive phase. These include short bowel syndrome, extensive small bowel inflammation, motility disorders, and deficiencies of digestive enzymes or bile salts. Diagnosis depends on symptoms, physical examination, and blood and stool tests. There is no gold standard for the diagnosis of malabsorption. Further testing should be based on the specific clinical context and the suspected underlying disease. Therapy is directed at nutritional support by enteral or parenteral feeding and screening for and supplementation of deficiencies in vitamins and minerals. Early enteral feeding is important for intestinal adaptation in short bowel syndrome. Medicinal treatment options for diarrhoea in malabsorption include loperamide, codeine, cholestyramine, or antibiotics. PMID:27086886

  16. 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

  17. Intestinal Obstruction

    MedlinePlus

    ... the small intestine (duodenum) may be caused by cancer of the pancreas, scarring from an ulcer, or Crohn disease . Rarely, a gallstone, a mass of undigested food, or a collection of parasitic worms may block ... commonly caused by cancer, diverticulitis , or a hard lump of stool (fecal ...

  18. Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

    DOE PAGESBeta

    Vandavasi, Venu Gopal; Weiss, Kevin L.; Cooper, Jonathan B.; Erskine, Peter T.; Tomanicek, Stephen J.; Ostermann, Andreas; Schrader, Tobias E.; Ginell, Stephan L.; Coates, Leighton

    2015-12-02

    The catalytic mechanism of class A beta-lactamases is often debated due in part to the large number of amino acids that interact with bound beta-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type beta-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 beta-lactamase with the antibiotic cefotaxime. The E166A mutant lacksmore » a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.« less

  19. Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

    SciTech Connect

    Vandavasi, Venu Gopal; Weiss, Kevin L.; Cooper, Jonathan B.; Erskine, Peter T.; Tomanicek, Stephen J.; Ostermann, Andreas; Schrader, Tobias E.; Ginell, Stephan L.; Coates, Leighton

    2015-12-02

    The catalytic mechanism of class A beta-lactamases is often debated due in part to the large number of amino acids that interact with bound beta-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type beta-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 beta-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.

  20. Construction of Global Acyl Lipid Metabolic Map by Comparative Genomics and Subcellular Localization Analysis in the Red Alga Cyanidioschyzon merolae

    PubMed Central

    Mori, Natsumi; Moriyama, Takashi; Toyoshima, Masakazu; Sato, Naoki

    2016-01-01

    Pathways of lipid metabolism have been established in land plants, such as Arabidopsis thaliana, but the information on exact pathways is still under study in microalgae. In contrast with Chlamydomonas reinhardtii, which is currently studied extensively, the pathway information in red algae is still in the state in which enzymes and pathways are estimated by analogy with the knowledge in plants. Here we attempt to construct the entire acyl lipid metabolic pathways in a model red alga, Cyanidioschyzon merolae, as an initial basis for future genetic and biochemical studies, by exploiting comparative genomics and localization analysis. First, the data of whole genome clustering by Gclust were used to identify 121 acyl lipid-related enzymes. Then, the localization of 113 of these enzymes was analyzed by GFP-based techniques. We found that most of the predictions on the subcellular localization by existing tools gave erroneous results, probably because these tools had been tuned for plants or green algae. The experimental data in the present study as well as the data reported before in our laboratory will constitute a good training set for tuning these tools. The lipid metabolic map thus constructed show that the lipid metabolic pathways in the red alga are essentially similar to those in A. thaliana, except that the number of enzymes catalyzing individual reactions is quite limited. The absence of fatty acid desaturation to produce oleic and linoleic acids within the plastid, however, highlights the central importance of desaturation and acyl editing in the endoplasmic reticulum, for the synthesis of plastid lipids as well as other cellular lipids. Additionally, some notable characteristics of lipid metabolism in C. merolae were found. For example, phosphatidylcholine is synthesized by the methylation of phosphatidylethanolamine as in yeasts. It is possible that a single 3-ketoacyl-acyl carrier protein synthase is involved in the condensation reactions of fatty acid

  1. Construction of Global Acyl Lipid Metabolic Map by Comparative Genomics and Subcellular Localization Analysis in the Red Alga Cyanidioschyzon merolae.

    PubMed

    Mori, Natsumi; Moriyama, Takashi; Toyoshima, Masakazu; Sato, Naoki

    2016-01-01

    Pathways of lipid metabolism have been established in land plants, such as Arabidopsis thaliana, but the information on exact pathways is still under study in microalgae. In contrast with Chlamydomonas reinhardtii, which is currently studied extensively, the pathway information in red algae is still in the state in which enzymes and pathways are estimated by analogy with the knowledge in plants. Here we attempt to construct the entire acyl lipid metabolic pathways in a model red alga, Cyanidioschyzon merolae, as an initial basis for future genetic and biochemical studies, by exploiting comparative genomics and localization analysis. First, the data of whole genome clustering by Gclust were used to identify 121 acyl lipid-related enzymes. Then, the localization of 113 of these enzymes was analyzed by GFP-based techniques. We found that most of the predictions on the subcellular localization by existing tools gave erroneous results, probably because these tools had been tuned for plants or green algae. The experimental data in the present study as well as the data reported before in our laboratory will constitute a good training set for tuning these tools. The lipid metabolic map thus constructed show that the lipid metabolic pathways in the red alga are essentially similar to those in A. thaliana, except that the number of enzymes catalyzing individual reactions is quite limited. The absence of fatty acid desaturation to produce oleic and linoleic acids within the plastid, however, highlights the central importance of desaturation and acyl editing in the endoplasmic reticulum, for the synthesis of plastid lipids as well as other cellular lipids. Additionally, some notable characteristics of lipid metabolism in C. merolae were found. For example, phosphatidylcholine is synthesized by the methylation of phosphatidylethanolamine as in yeasts. It is possible that a single 3-ketoacyl-acyl carrier protein synthase is involved in the condensation reactions of fatty acid

  2. Comparative studies of Acyl-CoA dehydrogenases for monomethyl branched chain substrates in amino acid metabolism.

    PubMed

    Liu, Xiaojun; Wu, Long; Deng, Guisheng; Chen, Gong; Li, Nan; Chu, Xiusheng; Li, Ding

    2013-04-01

    Short/branched chain acyl-CoA dehydrogenase (SBCAD), isovaleryl-CoA dehydrogenase (IVD), and isobutyryl-CoA dehydrogenase (IBD) are involved in metabolism of isoleucine, leucine, and valine, respectively. These three enzymes all belong to acyl-CoA dehydrogenase (ACD) family, and catalyze the dehydrogenation of monomethyl branched-chain fatty acid (mmBCFA) thioester derivatives. In the present work, the catalytic properties of rat SBCAD, IVD, and IBD, including their substrate specificity, isomerase activity, and enzyme inhibition, were comparatively studied. Our results indicated that SBCAD has its catalytic properties relatively similar to those of straight-chain acyl-CoA dehydrogenases in terms of their isomerase activity and enzyme inhibition, while IVD and IBD are different. IVD has relatively broader substrate specificity than those of the other two enzymes in accommodating various substrate analogs. The present study increased our understanding for the metabolism of monomethyl branched-chain fatty acids (mmBCFAs) and branched-chain amino acids (BCAAs), which should also be useful for selective control of a particular reaction through the design of specific inhibitors. PMID:23474214

  3. Acyl-ACP thioesterases from Camelina sativa: cloning, enzymatic characterization and implication in seed oil fatty acid composition.

    PubMed

    Rodríguez-Rodríguez, Manuel Fernando; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2014-11-01

    Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo fatty acid biosynthesis in the plastids of higher plants by hydrolyzing the thioester bond between ACP and the fatty acid synthesized. Free fatty acids are then esterified with coenzyme A prior to being incorporated into the glycerolipids synthesized through the eukaryotic pathway. Acyl-ACP thioesterases belong to the TE14 family of thioester-active enzymes and can be classified as FatAs and FatBs, which differ in their amino acid sequence and substrate specificity. Here, the FatA and FatB thioesterases from Camelina sativa seeds, a crop of interest in plant biotechnology, were cloned, sequenced and characterized. The mature proteins encoded by these genes were characterized biochemically after they were heterologously expressed in Escherichia coli and purified. C. sativa contained three different alleles of both the FatA and FatB genes. These genes were expressed most strongly in expanding tissues in which lipids are very actively synthesized, such as developing seed endosperm. The CsFatA enzyme displayed high catalytic efficiency on oleoyl-ACP and CsFatB acted efficiently on palmitoyl-ACP. The contribution of these two enzymes to the synthesis of C. sativa oil was discussed in the light of these results.

  4. Broad-range and binary-range acyl-acyl-carrier protein thioesterases suggest an alternative mechanism for medium-chain production in seeds.

    PubMed

    Voelker, T A; Jones, A; Cranmer, A M; Davies, H M; Knutzon, D S

    1997-06-01

    In the current model of medium-chain (C8-14) fatty acid biosynthesis in seeds, specialized FatB acyl-acyl-carrier-protein (ACP) thioesterases are responsible for the production of medium chains. We have isolated and characterized FatB cDNAs from the maturing seeds of elm (Ulmus americana) and nutmeg (Myristica fragrans), which accumulate predominantly caprate (10:0)- and myristate (14:0)-containing oils, respectively. In neither species were we able to find cDNAs encoding enzymes specialized for these chain lengths. Nutmeg FatB hydrolyses C14-18 substrates in vitro and expression in Brassica napus seeds leads to an oil enriched in C14-18 saturates. Elm FatB1 displays a binary specificity: one activity is centered on 10:0-ACP, and a second is centered on palmitate (16:0)-ACP. After expression in B. napus seeds the oil is enriched in C10-18 saturates, predominantly 16:0, 14:0, and 10:0. The composition of free fatty acids produced by elm FatB1 in Escherichia coli shifts from C14-16 to mostly C8-10 by increasing the rate of chain termination by this enzyme. These results suggest the existence of an alternative mechanism used in the evolution of medium-chain production, a model of which is presented. PMID:9193098

  5. 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

  6. Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase.

    PubMed

    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.

  7. Acylated iridoids with cytotoxicity from Valeriana jatamansi.

    PubMed

    Lin, Sheng; Shen, Yun-Heng; Li, Hui-Liang; Yang, Xian-Wen; Chen, Tao; Lu, Long-Hai; Huang, Zheng-Sheng; Liu, Run-Hui; Xu, Xi-Ke; Zhang, Wei-Dong; Wang, Hui

    2009-04-01

    Thirteen new acylated iridoids, jatamanvaltrates A-M (1-13), together with nine known valepotriates (14-22), were isolated from the whole plants of Valeriana jatamansi (syn. Valeriana wallichii). The structures of these new compounds were assigned by detailed interpretation of spectroscopic data. Jatamanvaltrates D (4) and H (9) are the first examples of naturally occurring valepotriates containing an o-hydroxybenzoyloxy moiety at C-10. All isolated compounds were tested for their cytotoxicity against lung adenocarcinoma (A549), metastatic prostate cancer (PC-3M), colon cancer (HCT-8), and hepatoma (Bel7402) cell lines.

  8. Intestinal spirochaetosis

    PubMed Central

    Lee, F. D.; Kraszewski, A.; Gordon, J.; Howie, J. G. R.; McSeveney, D.; Harland, W. A.

    1971-01-01

    An abnormal condition of the large intestine is described in which the surface epithelium is infested by short spirochaetes. Diagnosis can be made by light microscopy. A review of 14 cases diagnosed by rectal biopsy and 62 cases involving the appendix shows no consistent symptom complex. The possible significance is discussed. ImagesFig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 1 PMID:5548558

  9. Small intestinal ischemia and infarction

    MedlinePlus

    ... small intestine; Atherosclerosis - small intestine; Hardening of the arteries - small intestine ... Embolus: Blood clots can block one of the arteries supplying the intestine. People who have had a ...

  10. Secondary acylation of Klebsiella pneumoniae lipopolysaccharide contributes to sensitivity to antibacterial peptides.

    PubMed

    Clements, Abigail; Tull, Dedreia; Jenney, Adam W; Farn, Jacinta L; Kim, Sang-Hyun; Bishop, Russell E; McPhee, Joseph B; Hancock, Robert E W; Hartland, Elizabeth L; Pearse, Martin J; Wijburg, Odilia L C; Jackson, David C; McConville, Malcolm J; Strugnell, Richard A

    2007-05-25

    Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055DeltalpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055DeltalpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins.

  11. Role of acyl carrier protein isoforms in plant lipid metabolism: Progress report

    SciTech Connect

    Ohlrogge, J.B.

    1989-01-01

    Previous research from my lab has revealed that several higher plant species have multiple isoforms of acyl carrier protein (ACP) and therefore this trait appears highly conserved among higher plants. This level of conservation suggests that the existence of ACP isoforms is not merely the results of neutral gene duplications. We have developed techniques to examine a wider range of species. Acyl carrier proteins can be labelled very specifically and to high specific activity using H-palmitate and the E. coli enzyme acyl-ACP synthetase. Isoforms were then resolved by western blotting and native PAGE of H-palmitate labelled ACP's. Multiple isoforms of ACP were observed the leaf tissue of the monocots Avena sativa and Hordeum vulgare and dicots including Arabidopsis thallina, Cuphea wrightii, and Brassica napus. Lower vascular plants including the cycad, Dioon edule, Ginkgo biloba, the gymnosperm Pinus, the fern Anernia phyllitidis and Psilotum nudum, the most primitive known extant vascular plant, were also found to have multiple ACP isoforms as were the nonvascular liverwort, Marchantia and moss, Polytrichum. Therefore, the development of ACP isoforms occurred early in evolution. However, the uniellular alge Chlamydomonas and Dunaliella and the photosynthetic cyanobacteria Synechocystis and Agmnellum have only a single elecrophotetic form of ACP. Thus, multiple forms of ACP do not occur in all photosynthetic organisms but may be associated with multicellular plants.

  12. An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog

    SciTech Connect

    Van De Loo, F.J.; Broun, P.; Turner, S.; Somerville, C.

    1995-07-18

    Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with {approx}67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution. 26 refs., 6 figs., 1 tab.

  13. The role of Δ6-desaturase acyl-carrier specificity in the efficient synthesis of long-chain polyunsaturated fatty acids in transgenic plants.

    PubMed

    Sayanova, Olga; Ruiz-Lopez, Noemi; Haslam, Richard P; Napier, Johnathan A

    2012-02-01

    The role of acyl-CoA-dependent Δ6-desaturation in the heterologous synthesis of omega-3 long-chain polyunsaturated fatty acids was systematically evaluated in transgenic yeast and Arabidopsis thaliana. The acyl-CoA Δ6-desaturase from the picoalga Ostreococcus tauri and orthologous activities from mouse (Mus musculus) and salmon (Salmo salar) were shown to generate substantial levels of Δ6-desaturated acyl-CoAs, in contrast to the phospholipid-dependent Δ6-desaturases from higher plants that failed to modify this metabolic pool. Transgenic plants expressing the acyl-CoA Δ6-desaturases from either O. tauri or salmon, in conjunction with the two additional activities required for the synthesis of C20 polyunsaturated fatty acids, contained higher levels of eicosapentaenoic acid compared with plants expressing the borage phospholipid-dependent Δ6-desaturase. The use of acyl-CoA-dependent Δ6-desaturases almost completely abolished the accumulation of unwanted biosynthetic intermediates such as γ-linolenic acid in total seed lipids. Expression of acyl-CoA Δ6-desaturases resulted in increased distribution of long-chain polyunsaturated fatty acids in the polar lipids of transgenic plants, reflecting the larger substrate pool available for acylation by enzymes of the Kennedy pathway. Expression of the O. tauriΔ6-desaturase in transgenic Camelina sativa plants also resulted in the accumulation of high levels of Δ6-desaturated fatty acids. This study provides evidence for the efficacy of using acyl-CoA-dependent Δ6-desaturases in the efficient metabolic engineering of transgenic plants with high value traits such as the synthesis of omega-3 LC-PUFAs. PMID:21902798

  14. Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthases KasA and KasB.

    PubMed

    Schaeffer, M L; Agnihotri, G; Volker, C; Kallender, H; Brennan, P J; Lonsdale, J T

    2001-12-14

    Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two beta-ketoacyl-ACP synthase (KAS) enzymes of the M. tuberculosis FASII system. KasA and KasB were expressed in E. coli and purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use of M. tuberculosis AcpM, suggesting that structural differences between AcpM and E. coli ACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery. PMID:11600501

  15. Probing the Mechanism of the Mycobacterium tuberculosis [beta]-Ketoacyl-Acyl Carrier Protein Synthase III mtFabH: Factors Influencing Catalysis and Substrate Specificity

    SciTech Connect

    Brown, Alistair K.; Sridharan, Sudharsan; Kremer, Laurent; Lindenberg, Sandra; Dover, Lynn G.; Sacchettini, James C.; Besra, Gurdyal S.

    2010-11-30

    Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These {alpha}-alkyl, {beta}-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C{sub 24}-C{sub 26} fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The {beta}-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg{sup 46} revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg{sup 161} {yields} Ala substitution. Our structural studies suggested that His{sup 258}, previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys{sup 122}.

  16. Chlorsulfuron modifies biosynthesis of acyl Acid substituents of sucrose esters secreted by tobacco trichomes.

    PubMed

    Kandra, L; Wagner, G J

    1990-11-01

    Sucrose esters and duvatrienediol diterpenes are principal constituents formed in and secreted outside head cells of trichomes occurring on surfaces of Nicotiana tabacum. Using trichome-bearing epidermal peels prepared from midveins of N. tabacum cv T.I. 1068 leaves, we found that chlorsulfuron reduced and modified radiolabeling of sucrose ester acyl acids derived from branched-chain amino acid metabolism. The herbicide did not effect formation and exudation of diterpenes which are products of isoprenoid metabolism. Treatment with 1.0 micromolar chlorsulfuron affected 8.5- and 6.3-fold reductions in radiolabeling of methylvaleryl and methylbutyryl groups of sucrose esters, respectively, and concomitant increases of 9- and 9.8-fold in radiolabeling of straight chain valeryl and butyryl groups, respectively. These results and others indicate that inhibition of acetolactate synthase causes an accumulation of 2-oxo-butyric acid that is utilized by enzymes common to Leu biosynthesis to form 2-oxo-valeric acid. Coenzyme A (CoA) activation of this keto acid gives rise to butyryl CoA, which is utilized to form butyryl containing sucrose esters. Alternatively, reutilization of 2-oxo-valeric acid by the same enzymes followed by CoA activation leads to valeryl containing sucrose esters. We propose that in trichome secretory cells synthase, isomerase and dehydrogenase enzymes which catalyze Leu synthesis/degredation in most tissues, convert iso-branched, anteiso-branched and straight-chain keto acids in the formation of sucrose ester acyl groups. PMID:16667871

  17. Chlorsulfuron Modifies Biosynthesis of Acyl Acid Substituents of Sucrose Esters Secreted by Tobacco Trichomes

    PubMed Central

    Kandra, Lili; Wagner, George J.

    1990-01-01

    Sucrose esters and duvatrienediol diterpenes are principal constituents formed in and secreted outside head cells of trichomes occurring on surfaces of Nicotiana tabacum. Using trichome-bearing epidermal peels prepared from midveins of N. tabacum cv T.I. 1068 leaves, we found that chlorsulfuron reduced and modified radiolabeling of sucrose ester acyl acids derived from branched-chain amino acid metabolism. The herbicide did not effect formation and exudation of diterpenes which are products of isoprenoid metabolism. Treatment with 1.0 micromolar chlorsulfuron affected 8.5- and 6.3-fold reductions in radiolabeling of methylvaleryl and methylbutyryl groups of sucrose esters, respectively, and concomitant increases of 9- and 9.8-fold in radiolabeling of straight chain valeryl and butyryl groups, respectively. These results and others indicate that inhibition of acetolactate synthase causes an accumulation of 2-oxo-butyric acid that is utilized by enzymes common to Leu biosynthesis to form 2-oxo-valeric acid. Coenzyme A (CoA) activation of this keto acid gives rise to butyryl CoA, which is utilized to form butyryl containing sucrose esters. Alternatively, reutilization of 2-oxo-valeric acid by the same enzymes followed by CoA activation leads to valeryl containing sucrose esters. We propose that in trichome secretory cells synthase, isomerase and dehydrogenase enzymes which catalyze Leu synthesis/degredation in most tissues, convert iso-branched, anteiso-branched and straight-chain keto acids in the formation of sucrose ester acyl groups. PMID:16667871

  18. Emulsifying properties of acylated rapeseed (Brassica napus L.) peptides.

    PubMed

    Sánchez-Vioque, Raúl; Bagger, Christian L; Larré, Colette; Guéguen, Jacques

    2004-03-01

    A peptide fraction having an average size of 5.6 amino acids has been purified from a rapeseed hydrolyzate, acylated using C(10)-C(14) acyl chlorides, and the surface tension values at the air-water interface and emulsifying properties studied. As compared with standard surface-active proteins, such as bovine serum albumin (BSA), and with detergents such as sodium dodecyl sulfate (SDS), acylated peptides exhibited particular surface characteristics. The surface tension at air-water interface of acylated peptides ranged from 29.1 to 37.8 mN/m at equilibrium; these values were considerably lower than those for BSA and closer those for SDS, suggesting that acylated peptides pack at the air-water interface more like detergents than like proteins. The adsorption of acylated peptides to the oil-water interface was slower than for SDS or BSA, as deduced from the rather large size of oil droplets in emulsions (31-17 microm). Consequently, these emulsions creamed extensively during aging. Nevertheless, emulsions generated from acylated peptides were in general more stable to phase separation than those prepared from SDS. The C(14) acylated peptides were more effective for generating emulsions than the C(10) and C(12) derivatives, especially concerning the stability of emulsions against coalescence and phase separation, which was better than SDS and close to BSA.

  19. 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.

  20. 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…

  1. Biochemical characterization and substrate specificity of jojoba fatty acyl-CoA reductase and jojoba wax synthase.

    PubMed

    Miklaszewska, Magdalena; Banaś, Antoni

    2016-08-01

    Wax esters are used in industry for production of lubricants, pharmaceuticals and cosmetics. The only natural source of wax esters is jojoba oil. A much wider variety of industrial wax esters-containing oils can be generated through genetic engineering. Biotechnological production of tailor-made wax esters requires, however, a detailed substrate specificity of fatty acyl-CoA reductases (FAR) and wax synthases (WS), the two enzymes involved in wax esters synthesis. In this study we have successfully characterized the substrate specificity of jojoba FAR and jojoba WS. The genes encoding both enzymes were expressed heterologously in Saccharomyces cerevisiae and the activity of tested enzymes was confirmed by in vivo studies and in vitro assays using microsomal preparations from transgenic yeast. Jojoba FAR exhibited the highest in vitro activity toward 18:0-CoA followed by 20:1-CoA and 22:1-CoA. The activity toward other 11 tested acyl-CoAs was low or undetectable as with 18:2-CoA and 18:3-CoA. In assays characterizing jojoba WS combinations of 17 fatty alcohols with 14 acyl-CoAs were tested. The enzyme displayed the highest activity toward 14:0-CoA and 16:0-CoA in combination with C16-C20 alcohols as well as toward C18 acyl-CoAs in combination with C12-C16 alcohols. 20:1-CoA was efficiently utilized in combination with most of the tested alcohols.

  2. Interaction of GCAP1 with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation

    PubMed Central

    Peshenko, Igor V.; Olshevskaya, Elena V.; Dizhoor, Alexander M.

    2012-01-01

    Guanylyl cyclase activating proteins (GCAPs) are calcium/magnesium binding proteins within neuronal calcium sensor proteins group (NCS) of the EF-hand proteins superfamily. GCAPs activate retinal guanylyl cyclase (RetGC) in vertebrate photoreceptors in response to light-dependent fall of the intracellular free Ca2+ concentrations. GCAPs consist of four EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting N-myristoylation (Gly2 → Ala) on the ability of the recombinant GCAP1 to co-localize with its target enzyme when heterologously expressed in HEK293 cells. We also compared Ca2+ binding and RetGC-activating properties of the purified non-acylated G2A mutant and C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less effectively stimulated cyclase activity in vitro. Ca2+ binding isotherm of the G2A GCAP1 was slightly shifted toward higher free Ca2+ concentrations and so was Ca2+ sensitivity of RetGC reconstituted with the G2A mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand proximal to the myristoyl residue in three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism. PMID:22371697

  3. Diclofenac toxicity in human intestine ex vivo is not related to the formation of intestinal metabolites.

    PubMed

    Niu, Xiaoyu; de Graaf, Inge A M; Langelaar-Makkinje, Miriam; Horvatovich, Peter; Groothuis, Geny M M

    2015-01-01

    The use of diclofenac (DCF), a nonsteroidal anti-inflammatory drug, is associated with a high prevalence of gastrointestinal side effects. In vivo studies in rodents suggested that reactive metabolites of DCF produced by the liver or the intestine might be responsible for this toxicity. In the present study, precision-cut intestinal slices (PCIS) prepared from the jejunum of 18 human donors were used as an ex vivo model to investigate whether DCF intestinal metabolites are responsible for its intestinal toxicity in man. PCIS were incubated with a concentration range of DCF (0-600 µM) up to 24 h. DCF (≥400 µM) caused direct toxicity to the intestine as demonstrated by ATP depletion, morphological damage, caspase 3 activation, and lactate dehydrogenase leakage. Three main metabolites produced by PCIS (4'-hydroxy DCF, 5-hydroxy DCF, and DCF acyl glucuronide) were detected by HPLC. Protein adducts were detected by immunohistochemical staining and showed correlation with the intestinal metabolites. DCF induced similar toxicity to each of the samples regardless of the variation in metabolism among them. Less metabolites were produced by slices incubated with 400 µM DCF than with 100 µM DCF. The addition of the metabolic inhibitors such as ketoconazole, cimetidine, or borneol decreased the metabolite formation but increased the toxicity. The results suggest that DCF can induce intestinal toxicity in human PCIS directly at therapeutically relevant concentrations, independent of the reactive metabolites 4'-OH DCF, 5-OH DCF, or diclofenac acylglucuronide produced by the liver or formed in the intestine.

  4. Acyl peptidic siderophores: structures, biosyntheses and post-assembly modifications.

    PubMed

    Kem, Michelle P; Butler, Alison

    2015-06-01

    Acyl peptidic siderophores are produced by a variety of bacteria and possess unique amphiphilic properties. Amphiphilic siderophores are generally produced in a suite where the iron(III)-binding headgroup remains constant while the fatty acid appendage varies by length and functionality. Acyl peptidic siderophores are commonly synthesized by non-ribosomal peptide synthetases; however, the method of peptide acylation during biosynthesis can vary between siderophores. Following biosynthesis, acyl siderophores can be further modified enzymatically to produce a more hydrophilic compound, which retains its ferric chelating abilities as demonstrated by pyoverdine from Pseudomonas aeruginosa and the marinobactins from certain Marinobacter species. Siderophore hydrophobicity can also be altered through photolysis of the ferric complex of certain β-hydroxyaspartic acid-containing acyl peptidic siderophores. PMID:25677460

  5. Release of angiotensin converting enzyme-inhibitor peptides during in vitro gastrointestinal digestion of Parmigiano Reggiano PDO cheese and their absorption through an in vitro model of intestinal epithelium.

    PubMed

    Basiricò, L; Catalani, E; Morera, P; Cattaneo, S; Stuknytė, M; Bernabucci, U; De Noni, I; Nardone, A

    2015-11-01

    The occurrence of 8 bovine casein-derived peptides (VPP, IPP, RYLGY, RYLG, AYFYPEL, AYFYPE, LHLPLP, and HLPLP) reported as angiotensin converting enzyme-inhibitors (ACE-I) was investigated in the 3-kDa ultrafiltered water-soluble extract (WSE) of Parmigiano Reggiano (PR) cheese samples by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry via an electrospray ionization source. Only VPP, IPP, LHLPLP, and HLPLP were revealed in the WSE, and their total amount was in the range of 8.46 to 21.55 mg/kg of cheese. Following in vitro static gastrointestinal digestion, the same ACE-I peptides along with the newly formed AYFYPEL and AYFYPE were found in the 3 kDa WSE of PR digestates. Digestates presented high amounts (1,880-3,053 mg/kg) of LHLPLP, whereas the remaining peptides accounted for 69.24 to 82.82 mg/kg. The half-maximal inhibitory concentration (IC50) values decreased from 7.92 ± 2.08 in undigested cheese to 3.20 ± 1.69 after in vitro gastrointestinal digestion. The 3-kDa WSE of digested cheeses were used to study the transport of the 8 ACE-I peptides across the monolayers of the Caco-2 cell culture grown on a semipermeable membrane of the transwells. After 1h of incubation, 649.20 ± 148.85 mg/kg of LHLPLP remained in the apical compartment, whereas VPP, IPP, AYFYPEL, AYFYPE, and HLPLP accounted in total for less than 36.78 mg/kg. On average, 0.6% of LHLPLP initially present in the digestates added to the apical compartment were transported intact to the basolateral chamber after the same incubation time. Higher transport rate (2.9%) was ascertained for the peptide HLPLP. No other intact ACE-I peptides were revealed in the basolateral compartment. For the first time, these results demonstrated that the ACE-I peptides HLPLP and LHLPLP present in the in vitro digestates of PR cheese are partially absorbed through an in vitro model of human intestinal epithelium.

  6. K2CO3-promoted formation of aryl esters from primary aryl amides by the acyl-acyl exchange process.

    PubMed

    Bian, Yongjun; Qu, Xingyu

    2016-04-28

    A new acyl-acyl exchange reaction has been developed for the formation of aryl esters from primary aryl amides. The reaction could occur under mild reaction conditions with catalytic quantities of K2CO3, and could afford moderate to good yields of the desired products. PMID:27035611

  7. Vertebrate fatty acyl desaturase with Δ4 activity

    PubMed Central

    Li, Yuanyou; Monroig, Oscar; Zhang, Liang; Wang, Shuqi; Zheng, Xiaozhong; Dick, James R.; You, Cuihong; Tocher, Douglas R.

    2010-01-01

    Biosynthesis of the highly biologically active long-chain polyunsaturated fatty acids, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, in vertebrates requires the introduction of up to three double bonds catalyzed by fatty acyl desaturases (Fad). Synthesis of ARA is achieved by Δ6 desaturation of 18∶2n - 6 to produce 18∶3n - 6 that is elongated to 20∶3n - 6 followed by Δ5 desaturation. Synthesis of EPA from 18∶3n - 3 requires the same enzymes and pathway as for ARA, but DHA synthesis reportedly requires two further elongations, a second Δ6 desaturation and a peroxisomal chain shortening step. This paper describes cDNAs, fad1 and fad2, isolated from the herbivorous, marine teleost fish (Siganus canaliculatus) with high similarity to mammalian Fad proteins. Functional characterization of the cDNAs by heterologous expression in the yeast Saccharomyces cerevisiae showed that Fad1 was a bifunctional Δ6/Δ5 Fad. Previously, functional dual specificity in vertebrates had been demonstrated for a zebrafish Danio rerio Fad and baboon Fad, so the present report suggests bifunctionality may be more widespread in vertebrates. However, Fad2 conferred on the yeast the ability to convert 22∶5n - 3 to DHA indicating that this S. canaliculatus gene encoded an enzyme having Δ4 Fad activity. This is a unique report of a Fad with Δ4 activity in any vertebrate species and indicates that there are two possible mechanisms for DHA biosynthesis, a direct route involving elongation of EPA to 22∶5n - 3 followed by Δ4 desaturation, as well as the more complicated pathway as described above. PMID:20826444

  8. Friedel-Craft acylation of ar-himachalene: synthesis of acyl-ar-himachalene and a new acyl-hydroperoxide.

    PubMed

    Hossini, Issam; Harrad, Mohamed Anoir; Ait Ali, Mustapha; El Firdoussi, Larbi; Karim, Abdallah; Valerga, Pedro; Puerta, M Carmen

    2011-01-01

    Friedel-Craft acylation at 100 °C of 2,5,9,9-tetramethyl-6,7,8,9-tetrahydro-5H-benzocycloheptene [ar-himachalene], a sesquiterpenic hydrocarbon obtained by catalytic dehydrogenation of α-, β- and γ-himachalenes, produces a mixture of two compounds: (3,5,5,9-tetramethyl-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yl)-ethanone (2, in 69% yield), with a conserved reactant backbone, and 3, with a different skeleton, in 21% yield. The crystal structure of 3 reveals it to be 1-(8-ethyl-8-hydroperoxy-3,5,5-trimethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-ethanone. In this compound O-H…O bonds form dimers. These hydrogen-bonds, in conjunction with weaker C-H…O interactions, form a more extended supramolecular arrangement in the crystal. PMID:21760570

  9. Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

    SciTech Connect

    Serek, Robert; Forwood, Jade K.; Hume, David A.; Martin, Jennifer L.; Kobe, Bostjan

    2006-02-01

    The C-terminal domain of the mouse long-chain acyl-CoA thioesterase has been expressed in bacteria and crystallized by vapour diffusion. The crystals diffract to 2.4 Å resolution. The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination.

  10. Adipose triglyceride lipase is a TG hydrolase of the small intestine and regulates intestinal PPARα signaling.

    PubMed

    Obrowsky, Sascha; Chandak, Prakash G; Patankar, Jay V; Povoden, Silvia; Schlager, Stefanie; Kershaw, Erin E; Bogner-Strauss, Juliane G; Hoefler, Gerald; Levak-Frank, Sanja; Kratky, Dagmar

    2013-02-01

    Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating triglyceride (TG) hydrolysis. The lack of ATGL results in TG accumulation in multiple tissues, underscoring the critical role of ATGL in maintaining lipid homeostasis. Recent evidence suggests that ATGL affects TG metabolism via activation of peroxisome proliferator-activated receptor α (PPARα). To investigate specific effects of intestinal ATGL on lipid metabolism we generated mice lacking ATGL exclusively in the intestine (ATGLiKO). We found decreased TG hydrolase activity and increased intracellular TG content in ATGLiKO small intestines. Intragastric administration of [(3)H]trioleate resulted in the accumulation of radioactive TG in the intestine, whereas absorption into the systemic circulation was unchanged. Intraperitoneally injected [(3)H]oleate also accumulated within TG in ATGLiKO intestines, indicating that ATGL mobilizes fatty acids from the systemic circulation absorbed by the basolateral side from the blood. Down-regulation of PPARα target genes suggested modulation of cholesterol absorption by intestinal ATGL. Accordingly, ATGL deficiency in the intestine resulted in delayed cholesterol absorption. Importantly, this study provides evidence that ATGL has no impact on intestinal TG absorption but hydrolyzes TGs taken up from the intestinal lumen and systemic circulation. Our data support the role of ATGL in modulating PPARα-dependent processes also in the small intestine.

  11. Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

    PubMed Central

    Bowman, Thomas A.; O'Keeffe, Kayleigh R.; D'Aquila, Theresa; Yan, Qing Wu; Griffin, John D.; Killion, Elizabeth A.; Salter, Deanna M.; Mashek, Douglas G.; Buhman, Kimberly K.; Greenberg, Andrew S.

    2016-01-01

    Objective The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ACSL isoforms. In vitro studies have suggested a role for ACSL5 in triglyceride synthesis; however, we have limited understanding of the in vivo actions of this ACSL isoform. Methods To elucidate the in vivo actions of ACSL5 we generated a line of mice in which ACSL5 expression was ablated in all tissues (ACSL5−/−). Results Ablation of ACSL5 reduced ACSL activity by ∼80% in jejunal mucosa, ∼50% in liver, and ∼37% in brown adipose tissue lysates. Body composition studies revealed that ACSL5−/−, as compared to control ACSL5loxP/loxP, mice had significantly reduced fat mass and adipose fat pad weights. Indirect calorimetry studies demonstrated that ACSL5−/− had increased metabolic rates, and in the dark phase, increased respiratory quotient. In ACSL5−/− mice, fasting glucose and serum triglyceride were reduced; and insulin sensitivity was improved during an insulin tolerance test. Both hepatic mRNA (∼16-fold) and serum levels of fibroblast growth factor 21 (FGF21) (∼13-fold) were increased in ACSL5−/− as compared to ACSL5loxP/loxP. Consistent with increased FGF21 serum levels, uncoupling protein-1 gene (Ucp1) and PPAR-gamma coactivator 1-alpha gene (Pgc1α) transcript levels were increased in gonadal adipose tissue. To further evaluate ACSL5 function in intestine, mice were gavaged with an olive oil bolus; and the rate of triglyceride appearance in serum was found to be delayed in ACSL5−/− mice as compared to control mice. Conclusions In summary, ACSL5−/− mice have increased hepatic and serum FGF21 levels, reduced adiposity, improved insulin sensitivity, increased energy expenditure and delayed triglyceride absorption. These studies

  12. Characterization of Novel Acyl Coenzyme A Dehydrogenases Involved in Bacterial Steroid Degradation

    PubMed Central

    Ruprecht, Amanda; Maddox, Jaymie; Stirling, Alexander J.; Visaggio, Nicole

    2015-01-01

    ABSTRACT The acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) FadE34 and CasC, encoded by the cholesterol and cholate gene clusters of Mycobacterium tuberculosis and Rhodococcus jostii RHA1, respectively, were successfully purified. Both enzymes differ from previously characterized ACADs in that they contain two fused acyl-CoA dehydrogenase domains in a single polypeptide. Site-specific mutagenesis showed that only the C-terminal ACAD domain contains the catalytic glutamate base required for enzyme activity, while the N-terminal ACAD domain contains an arginine required for ionic interactions with the pyrophosphate of the flavin adenine dinucleotide (FAD) cofactor. Therefore, the two ACAD domains must associate to form a single active site. FadE34 and CasC were not active toward the 3-carbon side chain steroid metabolite 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4BNC-CoA) but were active toward steroid CoA esters containing 5-carbon side chains. CasC has similar specificity constants for cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA, while FadE34 has a preference for the last compound, which has a ring structure similar to that of cholesterol metabolites. Knockout of the casC gene in R. jostii RHA1 resulted in a reduced growth on cholate as a sole carbon source and accumulation of a 5-carbon side chain cholate metabolite. FadE34 and CasC represent unique members of ACADs with primary structures and substrate specificities that are distinct from those of previously characterized ACADs. IMPORTANCE We report here the identification and characterization of acyl-CoA dehydrogenases (ACADs) involved in the metabolism of 5-carbon side chains of cholesterol and cholate. The two homologous enzymes FadE34 and CasC, from M. tuberculosis and Rhodococcus jostii RHA1, respectively, contain two ACAD domains per polypeptide, and we show that these two domains interact to form a single active site. FadE34 and CasC are therefore representatives of a new class of

  13. Preparation of acetylsalicylic acid-acylated chitosan as a novel polymeric drug for drug controlled release.

    PubMed

    Liu, Changkun; Wu, Yiguang; Zhao, Liyan; Huang, Xinzheng

    2015-01-01

    The acetylsalicylic acid-acylated chitosan (ASACTS) with high degree of substitution (DS) was successfully synthesized, and characterized with FTIR, (1)H NMR and elemental analysis methods. The optimum synthesis conditions were obtained which gave the highest DS (about 60%) for ASACTS. Its drug release experiments were carried out in simulated gastric and intestine fluids. The results show that the drugs in the form of acetylsalicylic acid (ASA) and salicylic acid (SA) were released in a controlled manner from ASACTS only in simulated gastric fluid. The release profile can be best fitted with logistic and Weibull model. The research results reveal that ASACTS can be a potential polymeric drug for the controlled release of ASA and SA in the targeted gastric environment.

  14. Palladium-Catalyzed Environmentally Benign Acylation.

    PubMed

    Suchand, Basuli; Satyanarayana, Gedu

    2016-08-01

    Recent trends in research have gained an orientation toward developing efficient strategies using innocuous reagents. The earlier reported transition-metal-catalyzed carbonylations involved either toxic carbon monoxide (CO) gas as carbonylating agent or functional-group-assisted ortho sp(2) C-H activation (i.e., ortho acylation) or carbonylation by activation of the carbonyl group (i.e., via the formation of enamines). Contradicting these methods, here we describe an environmentally benign process, [Pd]-catalyzed direct carbonylation starting from simple and commercially available iodo arenes and aldehydes, for the synthesis of a wide variety of ketones. Moreover, this method comprises direct coupling of iodoarenes with aldehydes without activation of the carbonyl and also without directing group assistance. Significantly, the strategy was successfully applied to the synthesis n-butylphthalide and pitofenone. PMID:27377566

  15. Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592.

    PubMed Central

    Yan, R T; Chen, J S

    1990-01-01

    Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia. A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592. The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric. Purified ALDH contained no alcohol dehydrogenase activity. Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production. Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde. ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H). Kinetic data suggest a ping-pong mechanism for the reaction. ALDH was more stable in Tris buffer than in phosphate buffer. The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming). The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH. ALDH was O2 sensitive, but it could be protected against O2 inactivation by dithiothreitol. The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA in the presence or absence of dithiothreitol prior to assay. Images PMID:2275527

  16. Role of acyl carrier protein isoforms in plant lipid metabolism

    SciTech Connect

    Not Available

    1990-01-01

    Although acyl carrier protein (ACP) is the best studied protein in plant fatty acid biosynthesis, the in vivo forms of ACPs and their steady state pools have not been examined previously in either seed or leaf. Information about the relative pool sizes of free ACP and its acyl-ACP intermediates is essential for understanding regulation of de novo fatty acid biosynthesis in plants. In this study we utilized antibodies directed against spinach ACP as a sensitive assay to analyze the acyl groups while they were still covalently attached to ACPs. 4 refs., 4 figs.

  17. Sterol carrier protein2-like activity in rat intestine.

    PubMed

    Kharroubi, A; Wadsworth, J A; Chanderbhan, R; Wiesenfeld, P; Noland, B; Scallen, T; Vahouny, G V; Gallo, L L

    1988-03-01

    A sterol carrier protein2 (SCP2)-like activity has been demonstrated in rat intestinal mucosal homogenates and in isolated intestinal cells from both crypt and villus zones. The results indicate the presence of a protein with similar molecular weight and antigenicity to that of authentic SCP2 purified from rat liver cytosol. Like liver SCP2, mucosal cytosol stimulates pregnenolone production in rat adrenal mitochondria and acyl coenzyme A:cholesterol acyltransferase activity of liver and mucosal microsomes. The distribution of SCP2-like activity as determined by radioimmunoassay indicates high levels in mitochondria and cytosol and relatively lower levels in microsomes and in brush-border membranes. The widespread distribution of SCP2-like protein in the intestine is consistent with potential transfer functions in all phases of cholesterol processing. PMID:3379341

  18. Silencing the expression of mitochondrial acyl-CoA thioesterase I and acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis.

    PubMed

    Maloberti, Paula; Castilla, Rocío; Castillo, Fernanda; Cornejo Maciel, Fabiana; Mendez, Carlos F; Paz, Cristina; Podestá, Ernesto J

    2005-04-01

    Arachidonic acid and its lypoxygenated metabolites play a fundamental role in the hormonal regulation of steroidogenesis. Reduction in the expression of the mitochondrial acyl-CoA thioesterase (MTE-I) by antisense or small interfering RNA (siRNA) and of the arachidonic acid-preferring acyl-CoA synthetase (ACS4) by siRNA produced a marked reduction in steroid output of cAMP-stimulated Leydig cells. This effect was blunted by a permeable analog of cholesterol that bypasses the rate-limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The inhibition of steroidogenesis was overcome by addition of exogenous arachidonic acid, indicating that the enzymes are part of the mechanism responsible for arachidonic acid release involved in steroidogenesis. Knocking down the expression of MTE-I leads to a significant reduction in the expression of steroidogenic acute regulatory protein. This protein is induced by arachidonic acid and controls the rate-limiting step. Overexpression of MTE-I resulted in an increase in cAMP-induced steroidogenesis. In summary, our results demonstrate a critical role for ACS4 and MTE-I in the hormonal regulation of steroidogenesis as a new pathway of arachidonic acid release different from the classical phospholipase A2 cascade.

  19. Intestinal protozoa.

    PubMed

    Juckett, G

    1996-06-01

    Giardia is the best known cause of protozoal gastrointestinal disease in North America, producing significant but not life-threatening gastrointestinal distress and diarrhea. Although diagnosis of giardiasis may be challenging, treatment is usually successful. Entamoeba histolytica poses a rarer but far more difficult clinical challenge. Dysentery caused by E. histolytica may be the most feared intestinal protozoal infection, although Cryptosporidium parvum, Balantidium coli, Isospora belli, Sarcocystis species and other newly described protozoa also may cause diarrhea in healthy individuals and may result in intractable, life-threatening illness in patients with acquired immunodeficiency syndrome or other immunosuppressive diseases. Certain protozoa once considered relatively unimportant, such as Cryptosporidium, are now recognized as significant causes of morbidity even in the United States, since transmission readily occurs through contaminated water. PMID:8644565

  20. Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

    PubMed Central

    Casabon, Israël; Swain, Kendra; Crowe, Adam M.

    2014-01-01

    Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 105 ± 0.03 × 105 M−1 s−1) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 105 ± 0.1 × 105 M−1 s−1 and 3.2 × 105 ± 0.3 × 105 M−1 s−1, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and

  1. Specificities of the Acyl-Acyl Carrier Protein (ACP) Thioesterase and Glycerol-3-Phosphate Acyltransferase for Octadecenoyl-ACP Isomers (Identification of a Petroselinoyl-ACP Thioesterase in Umbelliferae).

    PubMed Central

    Dormann, P.; Frentzen, M.; Ohlrogge, J. B.

    1994-01-01

    This study was designed to address the question: How specific for double bond position and conformation are plant enzymes that act on oleoyl-acyl carrier protein (ACP)? Octadecenoyl-ACPs with cis double bonds at positions [delta]6, [delta]7, [delta]8, [delta]9, [delta]10, [delta]11, or [delta]12 and elaidyl (18:1[delta]9trans)-ACP were synthesized and used to characterize the substrate specificity of the acyl-ACP thioesterase and acyl-ACP:sn-glycerol-3-phosphate acyltransferase. The two enzymes were found to be specific for the [delta]9 position of the double bond. The thioesterase was highly specific for the [delta]9 cis conformation, but the transferase was almost equally active with the cis and the trans isomer of 18:1[delta]9-ACP. In plants such as the Umbelliferae species coriander (Coriandrum sativum L.) that accumulate petroselinic acid (18:1[delta]6cis) in their seed triacylglycerols, a high petroselinoyl-ACP thioesterase activity was found in addition to the oleoyl-ACP thioesterase. The two activities could be separated by anion-exchange chromatography, indicating that the petroselinoyl-ACP thioesterase is represented by a distinct polypeptide. PMID:12232130

  2. Role of intramitochondrial arachidonic acid and acyl-CoA synthetase 4 in angiotensin II-regulated aldosterone synthesis in NCI-H295R adrenocortical cell line.

    PubMed

    Mele, Pablo G; Duarte, Alejandra; Paz, Cristina; Capponi, Alessandro; Podestá, Ernesto J

    2012-07-01

    Although the role of arachidonic acid (AA) in angiotensin II (ANG II)- and potassium-stimulated steroid production in zona glomerulosa cells is well documented, the mechanism responsible for AA release is not fully described. In this study we evaluated the mechanism involved in the release of intramitochondrial AA and its role in the regulation of aldosterone synthesis by ANG II in glomerulosa cells. We show that ANG II and potassium induce the expression of acyl-coenzyme A (CoA) thioesterase 2 and acyl-CoA synthetase 4, two enzymes involved in intramitochondrial AA generation/export system well characterized in other steroidogenic systems. We demonstrate that mitochondrial ATP is required for AA generation/export system, steroid production, and steroidogenic acute regulatory protein induction. We also demonstrate the role of protein tyrosine phosphatases regulating acyl-CoA synthetase 4 and steroidogenic acute regulatory protein induction, and hence ANG II-stimulated aldosterone synthesis.

  3. 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

  4. The Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase from Acinetobacter sp. Strain ADP1: Characterization of a Novel Type of Acyltransferase

    PubMed Central

    Stöveken, Tim; Kalscheuer, Rainer; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

    2005-01-01

    The wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) catalyzes the final steps in triacylglycerol (TAG) and wax ester (WE) biosynthesis in the gram-negative bacterium Acinetobacter sp. strain ADP1. It constitutes a novel class of acyltransferases which is fundamentally different from acyltransferases involved in TAG and WE synthesis in eukaryotes. The enzyme was purified by a three-step purification protocol to apparent homogeneity from the soluble fraction of recombinant Escherichia coli Rosetta (DE3)pLysS (pET23a::atfA). Purified WS/DGAT revealed a remarkably low substrate specificity, accepting a broad range of various substances as alternative acceptor molecules. Besides having DGAT and WS activity, the enzyme possesses acyl-CoA:monoacylglycerol acyltransferase (MGAT) activity. The sn-1 and sn-3 positions of acylglycerols are accepted with higher specificity than the sn-2 position. Linear alcohols ranging from ethanol to triacontanol are efficiently acylated by the enzyme, which exhibits highest specificities towards medium-chain-length alcohols. The acylation of cyclic and aromatic alcohols, such as cyclohexanol or phenylethanol, further underlines the unspecific character of this enzyme. The broad range of possible substrates may lead to biotechnological production of interesting wax ester derivatives. Determination of the native molecular weight revealed organization as a homodimer. The large number of WS/DGAT-homologous genes identified in pathogenic mycobacteria and their possible importance for the pathogenesis and latency of these bacteria makes the purified WS/DGAT from Acinetobacter sp. strain ADP1 a valuable model for studying this group of proteins in pathogenic mycobacteria. PMID:15687201

  5. Effects of acute resistance exercise on acyl-ghrelin and obestatin levels in hemodialysis patients: a pilot study.

    PubMed

    Moraes, Cristiane; Borges, Natália A; Barboza, Jorge; Barros, Amanda F; Mafra, Denise

    2015-11-01

    Chronic physical exercises may be beneficial to modulate appetite hormones as acyl-ghrelin (orexigenic) and obestatin (anorexigenic) in chronic kidney disease (CKD) patients; however, there are no data about the effects of acute exercises on these hormones. Thus, the aim of the present study was to assess the effect of acute resistance exercise on appetite hormones (acyl-ghrelin and obestatin) of patients undergoing hemodialysis (HD). Twenty-five patients (44.7 ± 12.9 years, 68% women) on regular HD program were enrolled into two groups, 16 patients performed exercises and 9 patients comprised the control group. The patients performed the exercises in both lower limbs with ankle-cuffs and elastic bands, 30 min after the initiation of hemodialysis session. Blood samples of both the groups were drawn in the morning before and after 30 min with exercise session (exercise group) and, before and after the same time without exercise (control group). Acyl-ghrelin and obestatin plasma levels were measured using an enzyme immunometric assay. Acyl-ghrelin plasma levels did not change in both the groups. However, when stratified by gender the acyl-ghrelin increased significantly right after exercise in men [32.1 pg/mL (25.6-41.2) to 46.0 pg/mL (39.0-59.5)] (p = 0.04). Obestatin plasma levels reduced after a single bout of exercise and changes remained significantly when the sample was stratified by gender. There was no change in obestatin plasma levels in control group. A single bout of resistance exercise seems to modulate the levels of appetite hormones in HD patients. PMID:26381714

  6. Effects of acute resistance exercise on acyl-ghrelin and obestatin levels in hemodialysis patients: a pilot study.

    PubMed

    Moraes, Cristiane; Borges, Natália A; Barboza, Jorge; Barros, Amanda F; Mafra, Denise

    2015-11-01

    Chronic physical exercises may be beneficial to modulate appetite hormones as acyl-ghrelin (orexigenic) and obestatin (anorexigenic) in chronic kidney disease (CKD) patients; however, there are no data about the effects of acute exercises on these hormones. Thus, the aim of the present study was to assess the effect of acute resistance exercise on appetite hormones (acyl-ghrelin and obestatin) of patients undergoing hemodialysis (HD). Twenty-five patients (44.7 ± 12.9 years, 68% women) on regular HD program were enrolled into two groups, 16 patients performed exercises and 9 patients comprised the control group. The patients performed the exercises in both lower limbs with ankle-cuffs and elastic bands, 30 min after the initiation of hemodialysis session. Blood samples of both the groups were drawn in the morning before and after 30 min with exercise session (exercise group) and, before and after the same time without exercise (control group). Acyl-ghrelin and obestatin plasma levels were measured using an enzyme immunometric assay. Acyl-ghrelin plasma levels did not change in both the groups. However, when stratified by gender the acyl-ghrelin increased significantly right after exercise in men [32.1 pg/mL (25.6-41.2) to 46.0 pg/mL (39.0-59.5)] (p = 0.04). Obestatin plasma levels reduced after a single bout of exercise and changes remained significantly when the sample was stratified by gender. There was no change in obestatin plasma levels in control group. A single bout of resistance exercise seems to modulate the levels of appetite hormones in HD patients.

  7. 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.

  8. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making

    PubMed Central

    Tuladhar, Rubina; Lum, Lawrence

    2015-01-01

    The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms. PMID:25849923

  9. Acylated and Desacylated Ghrelin, Preptin, Leptin, and Nesfatin-1 Peptide Changes Related to the Body Mass Index

    PubMed Central

    Ozkan, Yusuf; Timurkan, Esra Suay; Sahin, İbrahim; Timurkan, Mustafa; Citil, Cihan; Kalayci, Mehmet; Yilmaz, Musa; Catak, Zekiye

    2013-01-01

    This study examines the levels of acylated and desacylated ghrelin, preptin, leptin, and nesfatin-1 peptide changes related to the body mass index (BMI). The subjects were allocated to 5 groups depending on their BMIs as follows: Group I (BMI <18.5 kg/m2); Group II (BMI 18.5–24.9 kg/m2); Group III (BMI 25–29.9 kg/m2); Group IV (BMI 30–39.9 kg/m2); Group V (BMI >40 kg/m2). Serum acylated and desacylated ghrelin, preptin, and leptin levels were measured by the enzyme-linked immunosorbent assay (ELISA) and nesfatin-1 was measured by the enzyme immunoassay (EIA). Desacylated ghrelin levels showed a gradual and statistically significant drop from Group I to Group V, while preptin and leptin levels exhibited a gradual and significant increase from Group I to Group IV. Serum nesfatin-1 levels gradually, but not significantly, increased from Group I to Group III and showed a significant decrease in Groups IV and V. In conclusion, leptin, preptin, and acylated ghrelin (AG) levels increased with higher BMI, whereas desacylated ghrelin (DAG) decreased and nesfatin-1 showed no clear relationship to BMI. PMID:24371438

  10. Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase

    PubMed Central

    Tran, Timothy H.; Hsiao, Yu-Shan; Jo, Jeanyoung; Chou, Chi-Yuan; Dietrich, Lars E.P.; Walz, Thomas; Tong, Liang

    2014-01-01

    Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds 1–6. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase (ACC) is a target for drug discovery against diabetes, cancer and other diseases 7–9. We report here the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kD), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active toward short- and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase (LCC). The holoenzyme is a homo-hexamer with molecular weight of 720 kD. The 3.0 Å crystal structure of Mycobacterium avium subspecies paratuberculosis LCC (MapLCC) holoenzyme revealed an architecture that is strikingly different compared to those of related biotin-dependent carboxylases 10,11. In addition, the domains of each monomer have no direct contacts with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources. PMID:25383525

  11. Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans.

    PubMed

    Zhang, Xinxing; Li, Kunhua; Jones, Rachel A; Bruner, Steven D; Butcher, Rebecca A

    2016-09-01

    Caenorhabditis elegans secretes ascarosides as pheromones to communicate with other worms and to coordinate the development and behavior of the population. Peroxisomal β-oxidation cycles shorten the side chains of ascaroside precursors to produce the short-chain ascaroside pheromones. Acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, have different side chain-length specificities and enable C. elegans to regulate the production of specific ascaroside pheromones. Here, we determine the crystal structure of the acyl-CoA oxidase 1 (ACOX-1) homodimer and the ACOX-2 homodimer bound to its substrate. Our results provide a molecular basis for the substrate specificities of the acyl-CoA oxidases and reveal why some of these enzymes have a very broad substrate range, whereas others are quite specific. Our results also enable predictions to be made for the roles of uncharacterized acyl-CoA oxidases in C. elegans and in other nematode species. Remarkably, we show that most of the C. elegans acyl-CoA oxidases that participate in ascaroside biosynthesis contain a conserved ATP-binding pocket that lies at the dimer interface, and we identify key residues in this binding pocket. ATP binding induces a structural change that is associated with tighter binding of the FAD cofactor. Mutations that disrupt ATP binding reduce FAD binding and reduce enzyme activity. Thus, ATP may serve as a regulator of acyl-CoA oxidase activity, thereby directly linking ascaroside biosynthesis to ATP concentration and metabolic state.

  12. Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans.

    PubMed

    Zhang, Xinxing; Li, Kunhua; Jones, Rachel A; Bruner, Steven D; Butcher, Rebecca A

    2016-09-01

    Caenorhabditis elegans secretes ascarosides as pheromones to communicate with other worms and to coordinate the development and behavior of the population. Peroxisomal β-oxidation cycles shorten the side chains of ascaroside precursors to produce the short-chain ascaroside pheromones. Acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, have different side chain-length specificities and enable C. elegans to regulate the production of specific ascaroside pheromones. Here, we determine the crystal structure of the acyl-CoA oxidase 1 (ACOX-1) homodimer and the ACOX-2 homodimer bound to its substrate. Our results provide a molecular basis for the substrate specificities of the acyl-CoA oxidases and reveal why some of these enzymes have a very broad substrate range, whereas others are quite specific. Our results also enable predictions to be made for the roles of uncharacterized acyl-CoA oxidases in C. elegans and in other nematode species. Remarkably, we show that most of the C. elegans acyl-CoA oxidases that participate in ascaroside biosynthesis contain a conserved ATP-binding pocket that lies at the dimer interface, and we identify key residues in this binding pocket. ATP binding induces a structural change that is associated with tighter binding of the FAD cofactor. Mutations that disrupt ATP binding reduce FAD binding and reduce enzyme activity. Thus, ATP may serve as a regulator of acyl-CoA oxidase activity, thereby directly linking ascaroside biosynthesis to ATP concentration and metabolic state. PMID:27551084

  13. Phospholipid profiling identifies acyl chain elongation as a ubiquitous trait and potential target for the treatment of lung squamous cell carcinoma

    PubMed Central

    Marien, Eyra; Meister, Michael; Muley, Thomas; del Pulgar, Teresa Gomez; Derua, Rita; Spraggins, Jeffrey M.; Van de Plas, Raf; Vanderhoydonc, Frank; Machiels, Jelle; Binda, Maria Mercedes; Dehairs, Jonas; Willette-Brown, Jami; Hu, Yinling; Dienemann, Hendrik; Thomas, Michael; Schnabel, Philipp A.; Caprioli, Richard M.; Lacal, Juan Carlos; Waelkens, Etienne; Swinnen, Johannes V.

    2016-01-01

    Lung cancer is the leading cause of cancer death. Beyond first line treatment, few therapeutic options are available, particularly for squamous cell carcinoma (SCC). Here, we have explored the phospholipidomes of 30 human SCCs and found that they almost invariably (in 96.7% of cases) contain phospholipids with longer acyl chains compared to matched normal tissues. This trait was confirmed using in situ 2D-imaging MS on tissue sections and by phospholipidomics of tumor and normal lung tissue of the L-IkkαKA/KA mouse model of lung SCC. In both human and mouse, the increase in acyl chain length in cancer tissue was accompanied by significant changes in the expression of acyl chain elongases (ELOVLs). Functional screening of differentially expressed ELOVLs by selective gene knockdown in SCC cell lines followed by phospholipidomics revealed ELOVL6 as the main elongation enzyme responsible for acyl chain elongation in cancer cells. Interestingly, inhibition of ELOVL6 drastically reduced colony formation of multiple SCC cell lines in vitro and significantly attenuated their growth as xenografts in vivo in mouse models. These findings identify acyl chain elongation as one of the most common traits of lung SCC discovered so far and pinpoint ELOVL6 as a novel potential target for cancer intervention. PMID:26862848

  14. Effect of a mutagenized acyl-ACP thioesterase FATA allele from sunflower with improved activity in tobacco leaves and Arabidopsis seeds.

    PubMed

    Moreno-Pérez, Antonio Javier; Venegas-Calerón, Mónica; Vaistij, Fabián E; Salas, Joaquin J; Larson, Tony R; Garcés, Rafael; Graham, Ian A; Martínez-Force, Enrique

    2014-03-01

    The substrate specificity of the acyl-acyl carrier protein (ACP) thioesterases significantly determines the type of fatty acids that are exported from plastids. Thus, designing acyl-ACP thioesterases with different substrate specificities or kinetic properties would be of interest for plant lipid biotechnology to produce oils enriched in specialty fatty acids. In the present work, the FatA thioesterase from Helianthus annuus was used to test the impact of changes in the amino acids present in the binding pocket on substrate specificity and catalytic efficiency. Amongst all the mutated enzymes studied, Q215W was especially interesting as it had higher specificity towards saturated acyl-ACP substrates and higher catalytic efficiency compared to wild-type H. annuus FatA. Null, wild type and high-efficiency alleles were transiently expressed in tobacco leaves to check their effect on lipid biosynthesis. Expression of active FatA thioesterases altered the composition of leaf triacylglycerols but did not alter total lipid content. However, the expression of the wild type and the high-efficiency alleles in Arabidopsis thaliana transgenic seeds resulted in a strong reduction in oil content and an increase in total saturated fatty acid content. The role and influence of acyl-ACP thioesterases in plant metabolism and their possible applications in lipid biotechnology are discussed.

  15. Amine, Alcohol and Phosphine Catalysts for Acyl Transfer Reactions

    NASA Astrophysics Data System (ADS)

    Spivey, Alan C.; Arseniyadis, Stellios

    An overview of the area of organocatalytic asymmetric acyl transfer processes is presented including O- and N-acylation. The material has been ordered according to the structural class of catalyst employed rather than reaction type with the intention to draw mechanistic parallels between the manner in which the various reactions are accelerated by the catalysts and the concepts employed to control transfer of chiral information from the catalyst to the substrates.

  16. Availability of Locally Synthesized and Systemic Antibodies in the Intestine

    PubMed Central

    Fubara, Ernest S.; Freter, Rolf

    1972-01-01

    The present studies are concerned with the parameters which control the appearance of locally synthesized or serum-derived antibodies in the intestine. The data show that intestinal antibody may be found in rabbits as well as in conventional or germfree mice after active immunization with Vibrio cholerae. However, a large fraction of the intestinal antibody in rabbits and conventional mice originated from the serum as indicated by (i) analysis of correlation between serum and intestinal antibody titers, and (ii) the occurrence of intestinal antibody after parenteral administration of antiserum. In contrast, only locally synthesized 11S immunoglobulin A antibody was detected in the intestine of actively immunized germfree mice. No intestinal antibody was demonstrable in germfree mice after parenteral injection of V. cholerae antiserum. With respect to the appearance of serum antibody in the intestine, the response of conventionalized (ex-germfree) mice was intermediate between that of rabbits or conventional mice and germfree mice. The availability of serum-derived coproantibody in germfree and conventional mice was related to the rates of intestinal degradation of serum antibody. When enzymes were removed by prior washing of intestinal segments, serum antibodies entered the intestine of germfree or conventional mice at similar rates. Rates of entry of serum antibodies into the lumen were comparable at different levels of the small intestine. The presence of a normal enteric flora appeared to protect intestinal antibody from degradation by lowering the concentration or activity of intestinal enzymes. The results are discussed in relation to the question of whether antibacterial immunity to cholera involves local or systemic mechanisms. Images PMID:4638499

  17. Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria

    SciTech Connect

    Halavaty, Andrei S.; Kim, Youngchang; Minasov, George; Shuvalova, Ludmilla; Dubrovska, Ievgeniia; Winsor, James; Zhou, Min; Onopriyenko, Olena; Skarina, Tatiana; Papazisi, Leka; Kwon, Keehwan; Peterson, Scott N.; Joachimiak, Andrzej; Savchenko, Alexei; Anderson, Wayne F.

    2012-10-01

    The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpS{sub SA}), Vibrio cholerae (AcpS{sub VC}) and Bacillus anthracis (AcpS{sub BA}) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS{sub BA} is emphasized because of the two 3′, 5′-adenosine diphosphate (3′, 5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′, 5′-ADP is bound as the 3′, 5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′, 5′-ADP–AcpS binary complexes. The position of the second 3′, 5′-ADP has never been described before. It is in close proximity to the first 3′, 5′-ADP and the ACP-binding site. The coordination of two ADPs in AcpS{sub BA} may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.

  18. Reciprocal Influence of Protein Domains in the Cold-Adapted Acyl Aminoacyl Peptidase from Sporosarcina psychrophila

    PubMed Central

    Parravicini, Federica; Natalello, Antonino; Papaleo, Elena; De Gioia, Luca; Doglia, Silvia Maria; Lotti, Marina; Brocca, Stefania

    2013-01-01

    Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic α/β-hydrolase domain and by an N-terminal β-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named α1-helix) in the cold-adapted enzyme from Sporosarcina psychrophila (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the α1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the β-propeller. The α1-helix is required for the stability of the intact protein, as in other enzymes of the same family; however in this psychrophilic enzyme only, it destabilizes the isolated β-propeller. A single charged residue (E10) in the α1-helix plays a major role for the stability of the whole structure. Overall, a strict interaction of the SpAAP domains seems to be mandatory for the preservation of their reciprocal structural integrity and may witness their co-evolution. PMID:23457536

  19. 3-Oxoacyl-(acyl-carrier protein) reductase from avocado (Persea americana) fruit mesocarp.

    PubMed Central

    Sheldon, P S; Kekwick, R G; Sidebottom, C; Smith, C G; Slabas, A R

    1990-01-01

    The NADPH-linked 3-oxoacyl-(acyl-carrier protein) (ACP) reductase (EC 1.1.1.100), also known as 'beta-ketoacyl-ACP reductase', has been purified from the mesocarp of mature avocado pears (Persea americana). The enzyme is inactivated by low ionic strength and low temperature. On SDS/PAGE under reducing conditions, purified 3-oxoacyl-ACP reductase migrated as a single polypeptide giving a molecular mass of 28 kDa. Gel-filtration chromatography gave an apparent native molecular mass of 130 kDa, suggesting that the enzyme is tetrameric. The enzyme is inactivated by dilution, but some protection is afforded by the presence of NADPH. Kinetic constants have been determined using synthetic analogues as well as the natural ACP substrate. It exhibits a broad pH optimum around neutrality. Phenylglyoxal inactivates the enzyme, and partial protection is given by 1 mM-NADPH. Antibodies have been raised against the protein, which were used to localize it using immunogold electron microscopy. It is localized in plastids. N-Terminal amino-acid-sequence analysis was performed on the enzyme, and it shows close structural similarity with cytochrome f. Internal amino-acid-sequence data, derived from tryptic peptides, shows similarity with the putative gene products encoded by the nodG gene from the nitrogen-fixing bacterium Rhizobium meliloti and the gra III act III genes from Streptomyces spp. Images Fig. 2. Fig. 5. Fig. 6. PMID:2244875

  20. Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase

    SciTech Connect

    Mogelson, S.; Lange, L.G.

    1984-08-28

    Fatty acyl ethyl esters arise from an esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A. This study was designed to purify the enzyme(s) in rabbit myocardium that catalyze(s) this reaction. Enzyme activity in homogenates of myocardium, as assayed by the rate of synthesis of ethyl (/sup 14/C)oleate from 0.4 mM (/sup 14/C)oleic acid and 0.2 M ethanol, was 31 nmol/ (g x h), and was recovered in the 48400g supernatant. This soluble ethyl ester synthase activity bound to DEAE-cellulose at pH 8, and elution with a NaCl gradient (0-0.25 M0 separated two enzyme activities accounting for 13 and 87% of recovered synthase activity. The major enzyme activity was purified over 5000-fold to homogeneity. Gel electrophoresis showed a single polypeptide with M/sub r/ 26,000, and gel permeation chromatography under nondenaturing conditions indicated a M/sub r/ of 50,000 for the active enzyme. Kinetic analyses indicated that greatest rates of synthesis were observed with unsaturated octadecanoic fatty acid substrates. K/sub m/'s for these fatty acids were essentially identical and equal to 0.2 mM; substrate specificity resulted from varying K/sub m/'s for methanol, ethanol, 1-propanol, and 1-butanol, while V/sub max/ was constant at approximately 1.5 nmol/(mg x s). The amino acid analysis of this synthase distinguishes it from typical cholesterol esterases. When the enzyme is maximally active with respect to ethyl ester synthesis, it does not hydrolyze cholesterol oleate. Fatty acid ethyl esters are synthesized in myocardium primarily by a soluble dimeric enzyme comprised of two nearly identical subunits which esterifies free fatty acids with ethanol to produce a nonoxidative metabolite.

  1. Enhancement of (stereo)selectivity in dynamic kinetic resolution using a core-shell nanozeolite@enzyme as a bi-functional catalyst.

    PubMed

    Wang, Wanlu; Li, Xiang; Wang, Zhoujun; Tang, Yi; Zhang, Yahong

    2014-08-28

    A core-shell nanozeolite@enzyme bi-functional catalyst is constructed, which greatly improves selectivity and stereoselectivity of products in dynamic kinetic resolution of aromatic secondary alcohols compared with mixed catalysts, especially those involving small acyl donors.

  2. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8

    SciTech Connect

    Bagautdinov, Bagautdin Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-05-01

    The crystal structure of 3-oxoacyl-(acyl-carrier protein) synthase II from T. thermophilus HB8 has been determined at 2.0 Å resolution and compared with the structures of β-keto-ACP synthases from other sources. The β-ketoacyl-(acyl carrier protein) synthases (β-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 Å resolution. The crystal is orthorhombic, space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 Å, and contains one homodimer in the asymmetric unit. The subunits adopt the well known α-β-α-β-α thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ‘open’ conformation of the Phe396 side chain.

  3. Regioselective Acylation of Diols and Triols: The Cyanide Effect.

    PubMed

    Peng, Peng; Linseis, Michael; Winter, Rainer F; Schmidt, Richard R

    2016-05-11

    Central topics of carbohydrate chemistry embrace structural modifications of carbohydrates and oligosaccharide synthesis. Both require regioselectively protected building blocks that are mainly available via indirect multistep procedures. Hence, direct protection methods targeting a specific hydroxy group are demanded. Dual hydrogen bonding will eventually differentiate between differently positioned hydroxy groups. As cyanide is capable of various kinds of hydrogen bonding and as it is a quite strong sterically nondemanding base, regioselective O-acylations should be possible at low temperatures even at sterically congested positions, thus permitting formation and also isolation of the kinetic product. Indeed, 1,2-cis-diols, having an equatorial and an axial hydroxy group, benzoyl cyanide or acetyl cyanide as an acylating agent, and DMAP as a catalyst yield at -78 °C the thermodynamically unfavorable axial O-acylation product; acyl migration is not observed under these conditions. This phenomenon was substantiated with 3,4-O-unproteced galacto- and fucopyranosides and 2,3-O-unprotected mannopyranosides. Even for 3,4,6-O-unprotected galactopyranosides as triols, axial 4-O-acylation is appreciably faster than O-acylation of the primary 6-hydroxy group. The importance of hydrogen bonding for this unusual regioselectivity could be confirmed by NMR studies and DFT calculations, which indicate favorable hydrogen bonding of cyanide to the most acidic axial hydroxy group supported by hydrogen bonding of the equatorial hydroxy group to the axial oxygen. Thus, the "cyanide effect" is due to dual hydrogen bonding of the axial hydroxy group which enhances the nucleophilicity of the respective oxygen atom, permitting an even faster reaction for diols than for mono-ols. In contrast, fluoride as a counterion favors dual hydrogen bonding to both hydroxy groups leading to equatorial O-acylation. PMID:27104625

  4. Site-specific S-Acylation of Influenza Virus Hemagglutinin

    PubMed Central

    Brett, Katharina; Kordyukova, Larisa V.; Serebryakova, Marina V.; Mintaev, Ramil R.; Alexeevski, Andrei V.; Veit, Michael

    2014-01-01

    S-Acylation of hemagglutinin (HA), the main glycoprotein of influenza viruses, is an essential modification required for virus replication. Using mass spectrometry, we have previously demonstrated specific attachment of acyl chains to individual acylation sites. Whereas the two cysteines in the cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to a cysteine positioned at the end of the transmembrane region (TMR). Here we analyzed recombinant viruses containing HA with exchange of conserved amino acids adjacent to acylation sites or with a TMR cysteine shifted to a cytoplasmic location to identify the molecular signal that determines preferential attachment of stearate. We first developed a new protocol for sample preparation that requires less material and might thus also be suitable to analyze cellular proteins. We observed cell type-specific differences in the fatty acid pattern of HA: more stearate was attached if human viruses were grown in mammalian compared with avian cells. No underacylated peptides were detected in the mass spectra, and even mutations that prevented generation of infectious virus particles did not abolish acylation of expressed HA as demonstrated by metabolic labeling experiments with [3H]palmitate. Exchange of conserved amino acids in the vicinity of an acylation site had a moderate effect on the stearate content. In contrast, shifting the TMR cysteine to a cytoplasmic location virtually eliminated attachment of stearate. Thus, the location of an acylation site relative to the transmembrane span is the main signal for stearate attachment, but the sequence context and the cell type modulate the fatty acid pattern. PMID:25349209

  5. Metabolomics of Apc Min/+ mice genetically susceptible to intestinal cancer

    PubMed Central

    2014-01-01

    Background To determine how diets high in saturated fat could increase polyp formation in the mouse model of intestinal neoplasia, Apc Min/+ , we conducted large-scale metabolome analysis and association study of colon and small intestine polyp formation from plasma and liver samples of Apc Min/+ vs. wild-type littermates, kept on low vs. high-fat diet. Label-free mass spectrometry was used to quantify untargeted plasma and acyl-CoA liver compounds, respectively. Differences in contrasts of interest were analyzed statistically by unsupervised and supervised modeling approaches, namely Principal Component Analysis and Linear Model of analysis of variance. Correlation between plasma metabolite concentrations and polyp numbers was analyzed with a zero-inflated Generalized Linear Model. Results Plasma metabolome in parallel to promotion of tumor development comprises a clearly distinct profile in Apc Min/+ mice vs. wild type littermates, which is further altered by high-fat diet. Further, functional metabolomics pathway and network analyses in Apc Min/+ mice on high-fat diet revealed associations between polyp formation and plasma metabolic compounds including those involved in amino-acids metabolism as well as nicotinamide and hippuric acid metabolic pathways. Finally, we also show changes in liver acyl-CoA profiles, which may result from a combination of Apc Min/+ -mediated tumor progression and high fat diet. The biological significance of these findings is discussed in the context of intestinal cancer progression. Conclusions These studies show that high-throughput metabolomics combined with appropriate statistical modeling and large scale functional approaches can be used to monitor and infer changes and interactions in the metabolome and genome of the host under controlled experimental conditions. Further these studies demonstrate the impact of diet on metabolic pathways and its relation to intestinal cancer progression. Based on our results, metabolic signatures

  6. Intestinal Folate Absorption

    PubMed Central

    Olinger, Edward J.; Bertino, Joseph R.; Binder, Henry J.

    1973-01-01

    These studies were designed to determine whether pteroylmonoglutamic acid (PGA) at physiologic concentrations is transported across the small intestine unaltered or is reduced and methylated to the circulating folate form (5-methyltetrahydrofolate [5-MeFH4]) during absorption. [3H]PGA was incubated in vitro on the mucosal side of rat jejunum. Of the folate transferred to the serosal side, the percent identified as 5-MeFH4 by DEAE-Sephadex chromtography was inversely related to the initial mucosa PGA concentration: at 7, 20, and 2,000 nM, 44%, 34%, and 2%, respectively, was converted to 5-MeFH4. In contrast, less than 4% of the folate transferred across ileal mucosa was 5-MeFH4 when the initial mucosa concentration was 20 nM. Specific activity of dihydrofolate (DHF) reductase, the enzyme responsible for converting PGA to tetrahydrofolic acid, was measured in villus homogenates and was significantly greater in the jejunum than in the ileum. 1,000 nM methotrexate (MTX), a DHF reductase inhibitor, markedly inhibited PGA conversion to 5-MeFH4 by the jejunum. Studies of transmural flux, initial rate of mucosal entry (influx) and mucosal accumulation (uptake) of folate were also performed. Although MTX did not alter the influx of PGA, MTX decreased jejunal mucosal uptake but increased transmural movement. Transmural folate movement across ileal mucosa was greater than across jejunal mucosa although mucosal uptake was greater in the jejunum than in the ileum. These results could explain previous studies which have failed to identify conversion of PGA to 5-MeFH4 when intestinal preparations have been exposed to higher and less physiologic concentrations of PGA. Further, these studies suggest that 5-MeFH4 may be retained by the jejunal mucosa. PMID:4727453

  7. Head-group acylation of monogalactosyldiacylglycerol is a common stress response, but the acyl-galactose acyl composition varies with the plant species and applied stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Head group acylation of monogalactosyldiacylglycerol is a plant lipid modification occurring during bacterial infection. Little is known about the range of stresses that induce this lipid modification, the molecular species induced, and the function of the modification. Lipidomic analysis using trip...

  8. Purification and properties of acyl/alkyl dihydroxyacetone-phosphate reductase from guinea pig liver peroxisomes.

    PubMed

    Datta, S C; Ghosh, M K; Hajra, A K

    1990-05-15

    The peroxisomal acyl/alkyl dihydroxyacetone-phosphate reductase (EC 1.1.1.101) was solubilized and purified 5500-fold from guinea pig liver. The enzyme could be solubilized by detergents only at high ionic strengths in presence of the cosubstrate NADPH. Peroxisomes, isolated from liver by a Nycodenz step density gradient centrifugation, were first treated with 0.2% Triton X-100 to remove the soluble and a large fraction of the membrane-bound proteins. The enzyme was solubilized from the resulting residue by 0.05% Triton X-100, 1 M KCl, 0.3 mM NADPH, and 2 mM dithiothreitol in Tris-HCl buffer (10 mM) at pH 7.5. The enzyme was further purified after precipitating it by dialyzing out the KCl and then resolubilized with 0.8% octyl glucoside in 1 M KCl (plus NADPH and dithiothreitol). The second solubilized enzyme was purified to homogeneity (370-fold from peroxisomes) by gel filtration in a Sepharose CL-6B column followed by affinity chromatography on an NADPH-agarose gel matrix. NADPH-agarose was prepared by reacting periodate-oxidized NADP+ to adipic acid dihydrazide-agarose and then reducing the immobilized NADP+ with NaBH4. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed a single homogeneous band with an apparent molecular weight of 60,000. The molecular weight of the native enzyme was estimated to be 75,000 by size exclusion chromatography. Amino acid analysis of the purified protein showed that hydrophobic amino acid comprised 27% of the molecule. The Km value of the purified enzyme for hexadecyldihydroxyacetone phosphate (DHAP) was 21 microM, and the Vmax value in the presence of 0.07 mM NADPH was 67 mumol/min/mg. The turnover number (Kcat), after correcting for the isotope effect of the cosubstrate NADP3H, was calculated to be 6,000 mol/min/mol of enzyme, assuming the enzyme has a molecular weight of 60,000. The purified enzyme also used palmitoyldihydroxyactone phosphate as a substrate (Km = 15.4 microM, and Vmax = 75

  9. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  10. Lipases in green chemistry: acylation and alcoholysis on steroids and nucleosides.

    PubMed

    Baldessari, Alicia; Iglesias, Luis E

    2012-01-01

    In this article, we describe the application of lipases in acylation and alcoholysis reactions on steroids and nucleosides. In the field of steroids, a variety of acetyl and fatty acid derivatives of androstanes, pregnanes, and cholestanes have been prepared through lipase-catalyzed acylation and alcoholysis reactions taking advantage of the high regio- and stereoselectivity of these enzymes. The substrates as well as the products show a high degree of biological activity as neurosteroids, hormones, and glucocorticoids. The regioselective preparation of diacylated nucleosides by means of an enzymatic alcoholysis allowed the synthesis of nucleosides prodrugs or modified nucleosides. The quantitative full deacylation and dealkoxycarbonylation of nucleosides and steroids is a mild synthetic method for the deprotection of these labile compounds. Some of the reported steroid and nucleoside products are novel, and it is not possible to obtain them satisfactorily by following traditional synthetic procedures. The advantages presented by this methodology, such as selectivity, mild reaction conditions, and low environmental impact, make the lipases an important tool in the application of the principles of Green Chemistry, offering a convenient way to prepare derivatives of natural compounds with a great potential in the pharmaceutical industry. PMID:22426734

  11. Accessibility of N-acyl-d-mannosamines to N-acetyl-d-neuraminic acid aldolase

    PubMed Central

    Pan, Yanbin; Ayani, Tiffany; Nadas, Janos; Wen, Shouming; Guo, Zhongwu

    2011-01-01

    N-Acetyl-d-neuraminic acid (NeuNAc) aldolase is an important enzyme for the metabolic engineering of cell surface NeuNAc using chemically modified d-mannosamines. To explore the optimal substrates for this application, eight N-acyl derivatives of d-mannosamine were prepared, and their accessibility to NeuNAc aldolase was investigated quantitatively. The N-propionyl-, N-butanoyl-, N-iso-butanoyl-, N-pivaloyl- and N-phenylacetyl-d-mannosamines proved to be as good substrates as, or even better than, the natural N-acetyl-d-mannosamine, while the N-trifluoropropionyl and benzoyl derivatives were poor. It was proposed that the electronic effects might have a significant influence on the enzymatic aldol condensation reaction of d-mannosamine derivatives, with electron-deficient acyl groups having a negative impact. The results suggest that N-propionyl-, N-butanoyl-, N-iso-butanoyl- and N-phenylacetyl-d-mannosamines may be employed to bioengineer NeuNAc on cells. PMID:15280054

  12. Probing the phosphopantetheine arm conformations of acyl carrier proteins using vibrational spectroscopy.

    PubMed

    Johnson, Matthew N R; Londergan, Casey H; Charkoudian, Louise K

    2014-08-13

    Acyl carrier proteins (ACPs) are universal and highly conserved domains central to both fatty acid and polyketide biosynthesis. These proteins tether reactive acyl intermediates with a swinging 4'-phosphopantetheine (Ppant) arm and interact with a suite of catalytic partners during chain transport and elongation while stabilizing the growing chain throughout the biosynthetic pathway. The flexible nature of the Ppant arm and the transient nature of ACP-enzyme interactions impose a major obstacle to obtaining structural information relevant to understanding polyketide and fatty acid biosynthesis. To overcome this challenge, we installed a thiocyanate vibrational spectroscopic probe on the terminal thiol of the ACP Ppant arm. This site-specific probe successfully reported on the local environment of the Ppant arm of two ACPs previously characterized by solution NMR, and was used to determine the solution exposure of the Ppant arm of an ACP from 6-deoxyerythronolide B synthase (DEBS). Given the sensitivity of the probe's CN stretching band to conformational distributions resolved on the picosecond time scale, this work lays a foundation for observing the dynamic action-related structural changes of ACPs using vibrational spectroscopy.

  13. Recognition of Acyl Carrier Proteins by Ketoreductases in Assembly Line Polyketide Synthases

    PubMed Central

    Ostrowski, Matthew P.; Cane, David E.; Khosla, Chaitan

    2016-01-01

    Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed a ca. 10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed a ca. 10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules. PMID:27118242

  14. Lipases in green chemistry: acylation and alcoholysis on steroids and nucleosides.

    PubMed

    Baldessari, Alicia; Iglesias, Luis E

    2012-01-01

    In this article, we describe the application of lipases in acylation and alcoholysis reactions on steroids and nucleosides. In the field of steroids, a variety of acetyl and fatty acid derivatives of androstanes, pregnanes, and cholestanes have been prepared through lipase-catalyzed acylation and alcoholysis reactions taking advantage of the high regio- and stereoselectivity of these enzymes. The substrates as well as the products show a high degree of biological activity as neurosteroids, hormones, and glucocorticoids. The regioselective preparation of diacylated nucleosides by means of an enzymatic alcoholysis allowed the synthesis of nucleosides prodrugs or modified nucleosides. The quantitative full deacylation and dealkoxycarbonylation of nucleosides and steroids is a mild synthetic method for the deprotection of these labile compounds. Some of the reported steroid and nucleoside products are novel, and it is not possible to obtain them satisfactorily by following traditional synthetic procedures. The advantages presented by this methodology, such as selectivity, mild reaction conditions, and low environmental impact, make the lipases an important tool in the application of the principles of Green Chemistry, offering a convenient way to prepare derivatives of natural compounds with a great potential in the pharmaceutical industry.

  15. Acyl Carrier Protein Synthases from Gram-Negative, Gram-Positive, and Atypical Bacterial Species: Biochemical and Structural Properties and Physiological Implications

    PubMed Central

    McAllister, Kelly A.; Peery, Robert B.; Zhao, Genshi

    2006-01-01

    Acyl carrier protein (ACP) synthase (AcpS) catalyzes the transfer of the 4′-phosphopantetheine moiety from coenzyme A (CoA) onto a serine residue of apo-ACP, resulting in the conversion of apo-ACP to the functional holo-ACP. The holo form of bacterial ACP plays an essential role in mediating the transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and phospholipids. AcpS is therefore an attractive target for therapeutic intervention. In this study, we have purified and characterized the AcpS enzymes from Escherichia coli, Streptococcus pneumoniae, and Mycoplasma pneumoniae, which exemplify gram-negative, gram-positive, and atypical bacteria, respectively. Our gel filtration column chromatography and cross-linking studies demonstrate that the AcpS enzyme from M. pneumoniae, like E. coli enzyme, exhibits a homodimeric structure, but the enzyme from S. pneumoniae exhibits a trimeric structure. Our biochemical studies show that the AcpS enzymes from M. pneumoniae and S. pneumoniae can utilize both short- and long-chain acyl CoA derivatives but prefer long-chain CoA derivatives as substrates. On the other hand, the AcpS enzyme from E. coli can utilize short-chain CoA derivatives but not the long-chain CoA derivatives tested. Finally, our biochemical studies show that M. pneumoniae AcpS is kinetically a very sluggish enzyme compared with those from E. coli and S. pneumoniae. Together, the results of these studies show that the AcpS enzymes from different bacterial species exhibit different native structures and substrate specificities with regard to the utilization of CoA and its derivatives. These findings suggest that AcpS from different microorganisms plays a different role in cellular physiology. PMID:16788183

  16. 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

  17. Two fatty acyl reductases involved in moth pheromone biosynthesis.

    PubMed

    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

  18. Small Intestine Disorders

    MedlinePlus

    Your small intestine is the longest part of your digestive system - about twenty feet long! It connects your stomach to ... many times to fit inside your abdomen. Your small intestine does most of the digesting of the foods ...

  19. Intestinal obstruction repair

    MedlinePlus

    Repair of volvulus; Intestinal volvulus - repair; Bowel obstruction - repair ... Intestinal obstruction repair is done while you are under general anesthesia . This means you are asleep and DO NOT feel pain. ...

  20. Large intestine (colon) (image)

    MedlinePlus

    ... portion of the digestive system most responsible for absorption of water from the indigestible residue of food. The ileocecal valve of the ileum (small intestine) passes material into the large intestine at the ...

  1. Vasoactive intestinal peptide test

    MedlinePlus

    ... medlineplus.gov/ency/article/003508.htm Vasoactive intestinal peptide test To use the sharing features on this page, please enable JavaScript. Vasoactive intestinal peptide (VIP) is a test that measures the amount ...

  2. Elucidation of the Mechanisms through Which the Reactive Metabolite Diclofenac Acyl Glucuronide Can Mediate Toxicity.

    PubMed

    Scialis, Renato J; Manautou, José E

    2016-04-01

    We have previously reported that mice lacking the efflux transporter Mrp3 had significant intestinal injury after toxic diclofenac (DCF) challenge, and proposed that diclofenac acyl glucuronide (DCF-AG), as a substrate of Mrp3, played a part in mediating injury. Since both humans and mice express the uptake transporter OATP2B1 in the intestines, OATP2B1 was characterized for DCF-AG uptake. In vitro assays using human embryonic kidney (HEK)-OATP2B1 cells demonstrated that DCF-AG was a substrate with a maximal velocity (Vmax) and Km of 17.6 ± 1.5 pmol/min per milligram and 14.3 ± 0.1 μM, respectively. Another key finding from our in vitro assays was that DCF-AG was more cytotoxic compared with DCF, and toxicity occurred within 1-3 hours of exposure. We also report that 1 mM DCF-AG caused a 6-fold increase in reactive oxygen species (ROS) by 3 hours. Investigation of oxidative stress through inhibition of superoxide dismutase (SOD) revealed that DCF-AG had 100% inhibition of SOD at the highest tested dose of 1 mM. The SOD and ROS results strongly suggest DCF-AG induced oxidative stress in vitro. Lastly, DCF-AG was screened for pharmacologic activity against COX-1 and COX-2 and was found to have IC50 values of 0.620 ± 0.105 and 2.91 ± 0.36 μM, respectively, which represents a novel finding. Since cyclooxygenase (COX) inhibition can lead to intestinal ulceration, it is plausible that DCF-AG can also contribute to enteropathy via COX inhibition. Taken in context, the work presented herein demonstrated the multifactorial pathways by which DCF-AG can act as a direct contributor to toxicity following DCF administration.

  3. Potential O-acyl-substituted (-)-Epicatechin gallate prodrugs as inhibitors of DMBA/TPA-induced squamous cell carcinoma of skin in Swiss albino mice.

    PubMed

    Vyas, Sandeep; Manon, Benu; Vir Singh, Tej; Dev Sharma, Pritam; Sharma, Manu

    2011-04-01

    (-)-Epicatechin-3-gallate (1) is one of the principal catechins of green tea and exhibits cancer-preventive activities in various animal models. However, this compound is unstable in neutral or alkaline medium and, therefore, has a poor bioavailability. To improve its stability, O-acyl derivatives of 1 were prepared by isolating the partially purified tea catechin fraction from green tea extract and treating it with a variety of acylating agents. The resulting derivatives, compounds 2-6, were screened for their antitumor potential against 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced squamous cell carcinogenesis of skin in mice. The results showed that the antitumor activity decreased with the increase in size of the chain length of the acyl groups, i.e., from compound 2, derivative with an Ac group, to compound 6, possessing a valeryl group. Moreover, the C(4) derivative with a branched acyl chain, 5, had a lower activity than the linear C(4) derivative 4. This reduction in the inhibitory activity may be due to the steric hindrance by the two Me groups. Moreover, significant increases in the protein levels analyzed by ELISA of c-Jun, p65, and p53 were observed in the skin of DMBA/TPA treated mice, whereas mice treated with 2 and DMBA/TPA had a similar expression of these transcription factors than the control mice. The prodrug potential of the O-acyl derivatives 2-6 showed that they were adequately stable to be absorbed intact from the intestine, more stable at gastric pH, and suitable for oral administration. PMID:21480506

  4. The halo-substituent effect on Pseudomonas cepacia lipase-mediated regioselective acylation of nucleosides: A comparative investigation.

    PubMed

    Wang, Zhao-Yu; Bi, Yan-Hong; Yang, Rong-Ling; Duan, Zhang-Qun; Nie, Ling-Hong; Li, Xiang-Qian; Zong, Min-Hua; Wu, Jie

    2015-10-20

    In this work, comparative experiments were explored to investigate the substrate specificity of Pseudomonas cepacia lipase in regioselective acylation of nucleosides carrying various substituents (such as the H, F, Cl, Br, I) at 2'- and 5-positions. Experimental data indicated that the catalytic performance of the enzyme depended very much on the halo-substituents in nucleosides. The increased bulk of 2'-substituents in ribose moiety of the nucleoside might contribute to the improved 3'-regioselectivity (90-98%, nucleosides a-d) in enzymatic decanoylation, while the enhancement of regioselectivity (93-99%) in 3'-O-acylated nucleosides e-h could be attributable to the increasing hydrophobicity of the halogen atoms at 5-positions. With regard to the chain-length selectivity, P. cepacia lipase displayed the highest 3'-regioselectivity toward the longer chain (C14) as compared to shorter (C6 and C10) ones. The position, orientation and property of the substituent, specific structure of the lipase's active site, and acyl structure could account for the diverse results. PMID:26325198

  5. Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress

    PubMed Central

    Hentchel, Kristy L.

    2015-01-01

    SUMMARY Acylation of biomolecules (e.g., proteins and small molecules) is a process that occurs in cells of all domains of life and has emerged as a critical mechanism for the control of many aspects of cellular physiology, including chromatin maintenance, transcriptional regulation, primary metabolism, cell structure, and likely other cellular processes. Although this review focuses on the use of acetyl moieties to modify a protein or small molecule, it is clear that cells can use many weak organic acids (e.g., short-, medium-, and long-chain mono- and dicarboxylic aliphatics and aromatics) to modify a large suite of targets. Acetylation of biomolecules has been studied for decades within the context of histone-dependent regulation of gene expression and antibiotic resistance. It was not until the early 2000s that the connection between metabolism, physiology, and protein acetylation was reported. This was the first instance of a metabolic enzyme (acetyl coenzyme A [acetyl-CoA] synthetase) whose activity was controlled by acetylation via a regulatory system responsive to physiological cues. The above-mentioned system was comprised of an acyltransferase and a partner deacylase. Given the reversibility of the acylation process, this system is also referred to as reversible lysine acylation (RLA). A wealth of information has been obtained since the discovery of RLA in prokaryotes, and we are just beginning to visualize the extent of the impact that this regulatory system has on cell function. PMID:26179745

  6. Dietary acylated starch improves performance and gut health in necrotic enteritis challenged broilers.

    PubMed

    M'Sadeq, Shawkat A; Wu, Shu-Biao; Swick, Robert A; Choct, Mingan

    2015-10-01

    Resistant starch has been reported to act as a protective agent against pathogenic organisms in the gut and to encourage the proliferation of beneficial organisms. This study examined the efficacy of acetylated high amylose maize starch (SA) and butyralated high-amylose maize starch (SB) in reducing the severity of necrotic enteritis (NE) in broilers under experimental challenge. A total of 720 one-day-old male Ross 308 chicks were assigned to 48 floor pens with a 2 × 4 factorial arrangement of treatments. Factors were a) challenge: no or yes; and b) feed additive: control, antibiotics (AB), SA, or SB. Birds were challenged with Eimeria and C. perfringens according to a previously reported protocol. On d 24 and 35, challenged birds had lower (P < 0.001) livability (LV), weight gain (WG), and feed intake (FI) compared to unchallenged birds. Challenged birds fed SA and SB had higher FI and WG at d 24 and 35 (P < 0.05) compared to birds fed the control diet, while being significantly lower than those fed AB. Unchallenged birds fed SA or SB had higher FI at d 24 and 35 compared to those fed the control diet (P < 0.05). Birds fed SB had increased (P < 0.001) jejunal villus height/crypt depth (VH:CD) ratios at d 15, increased ileal (P < 0.001) and caecal (P < 0.001) butyrate levels at d 15 and 24, and decreased (P < 0.01) caecal pH at d 15. Birds fed SA had increased (P < 0.001) ileal acetate content at d 24 and decreased (P < 0.01) caecal pH at d 15. These results demonstrated that dietary acylated starch improved WG in birds challenged with necrotic enteritis. Depending on the acid used, starch acylation also offers a degree of specificity in short chain fatty acid (SCFA) delivery to the lower intestinal tract which improves gut health.

  7. Chemical exposure and intestinal function.

    PubMed Central

    Schiller, C M

    1979-01-01

    The particular substances that are ingested by individuals are the consequence of their environmental, residential, and occupational exposures. The possible effects of these exposures on intestinal functions can be examined by the evaluation of in vivo or in vitro exposure followed by an in vivo and/or in vitro monitoring of effects. Several examples of the in vivo exposure and in vitro monitoring approach are presented to demonstrate the consequences of oral exposure to either a heavy metal (arsenic), or a herbicide contaminant (2,3,7,8-tetrachlorodibenzo-p-dioxin) or a jet fuel propellant (hydrazine) and the subsequent measurement of either a particular metabolic pathway, or a cell-specific enzyme induction or the development of brush border enzymes are presented. Images FIGURE 6. FIGURE 7. FIGURE 8. a FIGURE 8. b FIGURE 9. PMID:120255

  8. Identification of Unusual Phospholipid Fatty Acyl Compositions of Acanthamoeba castellanii

    PubMed Central

    Palusinska-Szysz, Marta; Kania, Magdalena; Turska-Szewczuk, Anna; Danikiewicz, Witold; Russa, Ryszard; Fuchs, Beate

    2014-01-01

    Acanthamoeba are opportunistic protozoan pathogens that may lead to sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba followed by aggressive treatment regimen. The plasma membrane of Acanthamoeba consists of 25% phospholipids (PL). The presence of C20 and, recently reported, 28- and 30-carbon fatty acyl residues is characteristic of amoeba PL. A detailed knowledge about this unusual PL composition could help to differentiate Acanthamoeba from other parasites, e.g. bacteria and develop more efficient treatment strategies. Therefore, the detailed PL composition of Acanthamoeba castellanii was investigated by 31P nuclear magnetic resonance spectroscopy, thin-layer chromatography, gas chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry. Normal and reversed phase liquid chromatography coupled with mass spectrometric detection was used for detailed characterization of the fatty acyl composition of each detected PL. The most abundant fatty acyl residues in each PL class were octadecanoyl (18∶0), octadecenoyl (18∶1 Δ9) and hexadecanoyl (16∶0). However, some selected PLs contained also very long fatty acyl chains: the presence of 28- and 30-carbon fatty acyl residues was confirmed in phosphatidylethanolamine (PE), phosphatidylserine, phosphatidic acid and cardiolipin. The majority of these fatty acyl residues were also identified in PE that resulted in the following composition: 28∶1/20∶2, 30∶2/18∶1, 28∶0/20∶2, 30∶2/20∶4 and 30∶3/20∶3. The PL of amoebae are significantly different in comparison to other cells: we describe here for the first time unusual, very long chain fatty acids with Δ5-unsaturation (30∶35,21,24) and 30∶221,24 localized exclusively in specific phospholipid classes of A. castellanii protozoa that could serve as specific biomarkers for the presence of these

  9. Increased prevalence of intestinal inflammation in patients with liver cirrhosis

    PubMed Central

    Saitoh, Osamu; Sugi, Kazunori; Kojima, Keishi; Matsumoto, Hisashi; Nakagawa, Ken; Kayazawa, Masanobu; Tanaka, Seigou; Teranishi, Tsutomu; Hirata, Ichiro; Katsu, Ken-ichi

    1999-01-01

    AIM: To investigate the pathophysiology of the digestive tract in patients with liver cirrhosis. METHODS: In 42 cirrhotic patients and 20 control subjects, the following fecal proteins were measured by enzyme-linked immunosorbent assay: albumin (Alb), transferrin (Tf), and α1-antitrypsin (α1-AT) as a marker for intestinal protein loss, hemoglobin (Hb) for bleeding, PMN-elastase for intestinal inflammation, and secretory IgA for intestinal immunity. RESULTS: The fecal concentrations of Hb, Alb, Tf, α1-AT, an d PMN-elastase were increased in 13 (31%), 8 (19%), 10 (24%), 6 (14%), and 11 (26%) cases among 42 patients, respectively. Fecal concentration of secretory IgA was decreased in 7 (17%) of 42 patients. However, these fecal concentrations were not related to the severity or etiology of liver cirrhosis. The serum Alb level was significantly decreased in patients with intestinal protein loss compared to that in patients without intestinal protein loss. CONCLUSION: These findings suggest that: ① besides the well-known pathological conditions, such as bleeding and protein loss, intestinal inflammation and decreased intestinal immunity are found in cirrhotic patients; ② intestinal protein loss contributes to hypoalbuminemia in cirrhotic patients, and ③ intestinal inflammation should not be over looked in cirrhotic patients, since it may contribute to or cause intestinal protein loss and other various path ological conditions. PMID:11819475

  10. Neural regulation of intestinal nutrient absorption.

    PubMed

    Mourad, Fadi H; Saadé, Nayef E

    2011-10-01

    The nervous system and the gastrointestinal (GI) tract share several common features including reciprocal interconnections and several neurotransmitters and peptides known as gut peptides, neuropeptides or hormones. The processes of digestion, secretion of digestive enzymes and then absorption are regulated by the neuro-endocrine system. Luminal glucose enhances its own absorption through a neuronal reflex that involves capsaicin sensitive primary afferent (CSPA) fibres. Absorbed glucose stimulates insulin release that activates hepatoenteric neural pathways leading to an increase in the expression of glucose transporters. Adrenergic innervation increases glucose absorption through α1 and β receptors and decreases absorption through activation of α2 receptors. The vagus nerve plays an important role in the regulation of diurnal variation in transporter expression and in anticipation to food intake. Vagal CSPAs exert tonic inhibitory effects on amino acid absorption. It also plays an important role in the mediation of the inhibitory effect of intestinal amino acids on their own absorption at the level of proximal or distal segment. However, chronic extrinsic denervation leads to a decrease in intestinal amino acid absorption. Conversely, adrenergic agonists as well as activation of CSPA fibres enhance peptides uptake through the peptide transporter PEPT1. Finally, intestinal innervation plays a minimal role in the absorption of fat digestion products. Intestinal absorption of nutrients is a basic vital mechanism that depends essentially on the function of intestinal mucosa. However, intrinsic and extrinsic neural mechanisms that rely on several redundant loops are involved in immediate and long-term control of the outcome of intestinal function.

  11. Intestinal lymphangiectasia secondary to radiotherapy and chemotherapy

    SciTech Connect

    Rao, S.S.; Dundas, S.; Holdsworth, C.D.

    1987-08-01

    We report a case of intestinal lymphangiectasia secondary to radiotherapy and chemotherapy. The patient also had small bowel bacterial overgrowth and pancreatic insufficiency. Lymphatic ectasia as a histological feature has been described previously in association with postradiotherapy malabsorption, but radiation-induced lymphangiectasia producing clinical manifestations has hitherto not been reported. Replacement of dietary long-chain fats with medium-chain triglycerides, pancreatic enzyme supplements, and a short course of oxytetracycline, resulted in dramatic clinical improvement. The possibility of intestinal lymphangiectasia should be borne in mind in patients with postradiotherapy malabsorption. A low serum albumin and lymphocyte count should draw attention to this possibility.

  12. Vertebrate Intestinal Endoderm Development

    PubMed Central

    Spence, Jason R.; Lauf, Ryan; Shroyer, Noah F.

    2010-01-01

    The endoderm gives rise to the lining of the esophagus, stomach and intestines, as well as associated organs. To generate a functional intestine, a series of highly orchestrated developmental processes must occur. In this review, we attempt to cover major events during intestinal development from gastrulation to birth, including endoderm formation, gut tube growth and patterning, intestinal morphogenesis, epithelial reorganization, villus emergence as well as proliferation and cytodifferentiation. Our discussion includes morphological and anatomical changes during intestinal development as well as molecular mechanisms regulating these processes. PMID:21246663

  13. 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

    SciTech Connect

    Vandevoorde, Severine; Saha, Bijali; Mahadevan, Anu; Razdan, Raj K.; Pertwee, Roger G.; Martin, Billy R.; Fowler, Christopher J. . E-mail: cf@pharm.umu.se

    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 (IC{sub 50} values in the range 5.1-8.2 {mu}M), whereas the two compounds with a single unsaturated bond were less potent (IC{sub 50} values 19 and 21 {mu}M). 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 IC{sub 50} values of 12 and 32 {mu}M, respectively. The 22-carbon chain analogue of 1-AG was also potent (IC{sub 50} value 4.5 {mu}M). 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.

  14. Mutation of active site serine residue with cysteine displays change in acyl-acceptor preference of β-peptidyl aminopeptidase from Pseudomonas aeruginosa PAO1.

    PubMed

    Arima, Jiro; Tanaka, Ayumi; Morimoto, Masazumi; Mori, Nobuhiro

    2014-02-01

    A β-peptidyl aminopeptidase, a peptidase belonging to the P1 family, catalyzes aminolysis in accordance with its hydrolytic activity. We specifically examined β-peptidyl aminopeptidase of Pseudomonas aeruginosa PAO1 (BapF) to assess the effects of mutation of catalytic Ser with Cys or Thr on its catalytic ability. Recombinant BapF and its S237C mutant exhibited p-nitroaniline release activity toward β-homo-Gly-p-nitroanilide (βhGly-pNA), but the products of the enzyme reaction differed completely from one another. Wild-type BapF showed βhGly-βhGly-pNA synthetic activity, but the product vanished in a few minutes and converted to free βhGly. In contrast, the product βhGly-βhGly-pNA was synthesized by S237C BapF efficiently without degradation, indicating that because of the mutation, the enzyme came to recognize only the amine group as an acyl acceptor instead of water. Furthermore, a difference in acyl acceptor preference between that of wild type and S237C BapF was observed. When using cysteamine as an acyl acceptor, βhGly-cysteamine was synthesized only in the reaction using S237C BapF. In contrast, S237C BapF was unable to synthesize βhGly-cystamine when using cystamine as an acyl acceptor, although it was synthesized by wild-type BapF. Such a dynamic change in the acyl acceptor by the mutation of catalytic Ser with Cys is regarded as a unique feature of family P1 peptidases.

  15. RhlA converts beta-hydroxyacyl-acyl carrier protein intermediates in fatty acid synthesis to the beta-hydroxydecanoyl-beta-hydroxydecanoate component of rhamnolipids in Pseudomonas aeruginosa.

    PubMed

    Zhu, Kun; Rock, Charles O

    2008-05-01

    Pseudomonas aeruginosa secretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the beta-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of beta-hydroxydecanoyl-beta-hydroxydecanoate from two molecules of beta-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily beta-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity in P. aeruginosa increases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for beta-hydroxydecanoyl-ACP. In Escherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.

  16. Pyripyropenes, novel inhibitors of acyl-CoA:cholesterol acyltransferase produced by Aspergillus fumigatus. I. Production, isolation, and biological properties.

    PubMed

    Tomoda, H; Kim, Y K; Nishida, H; Masuma, R; Omura, S

    1994-02-01

    Aspergillus fumigatus FO-1289, a soil isolate, was found to produce a series of novel inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). Four active compounds, named pyripyropenes A, B, C and D, were isolated from the fermentation broth of the producing strain by solvent extraction, silica gel column chromatography, ODS column chromatography and preparative HPLC. Pyripyropenes A, B, C and D show very potent ACAT inhibitory activity in an enzyme assay system using rat liver microsomes with IC50 values of 58, 117, 53 and 268 nM, respectively. PMID:8150709

  17. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  18. Synthesis, crystal structure, and in vitro and in silico molecular docking of novel acyl thiourea derivatives

    NASA Astrophysics Data System (ADS)

    Haribabu, Jebiti; Subhashree, Govindarajulu Rangabashyam; Saranya, Sivaraj; Gomathi, Kannayiram; Karvembu, Ramasamy; Gayathri, Dasararaju

    2015-08-01

    In the present study, a series of six biologically active substituted acyl thiourea compounds (1-6) has been synthesized from cyclohexanecarbonyl isothiocyanate and various primary amines (2-methyl aniline, aniline, 4-methoxy aniline, 4-ethoxy aniline, benzyl amine and 2-methoxy aniline). The synthesized compounds were characterized by elemental analyses, UV-Visible, FT-IR, 1H & 13C NMR and mass spectroscopic techniques. Three dimensional molecular structure of two compounds (1 and 5) was determined by single crystal X-ray crystallography. All the synthesized compounds show good anti-oxidant and anti-haemolytic activities. In silico molecular docking studies were performed to screen against DprE1 and HSP90 enzymes targeting tuberculosis and cancer respectively.

  19. An esterase on the outer membrane of Pseudomonas aeruginosa for the hydrolysis of long chain acyl esters.

    PubMed

    Ohkawa, I; Shiga, S; Kageyama, M

    1979-09-01

    A new esterase activity which hydrolyzes palmitoyl-CoA was found in the membrane fraction of Pseudomonas aeruginosa. All the 11 strains of P. aeruginosa tested possessed this esterase activity. The esterase was constitutive and was fully active on the intact cell bodies toward substrates in the medium. It was located on the outer membrane of the cell envelope, and was not released into the culture medium. This activity was designated as OM (outer membrane) esterase. OM esterase was solubilized from the cell envelope with EDTA-Triton X-100 and purified 690-fold. It was a minor component of the outer membrane. Its molecular weight was approximately 55,000. The activity was rather stable to heat, a wide range of pH, and treatment with detergents and organic solvents. No cofactors were required. The pH optimum of the reaction was 8.5. Among various acyl-CoAs, only long chain (C12--C18) thioesters were hydrolyzed. OM esterase also hydrolyzed some kinds of oxy-esters such as p-nitrophenyl acyl esters, monoacyl esters of sucrose and Tween 80 (polyoxyethylene sorbitan monooleate). On the other hand, triglycerides, phospholipids, or hydrophobic monoesters were not hydrolyzed at all. Thus, this enzyme seems to have specificity for long chain acyl esters with hydrophilic groups, whether thio- or oxy-ester. Mutants deficient in this esterase activity were isolated. These mutants were unable to grow on Tween 80 as a sole carbon source. This suggests a possible role of OM esterase in the utilization of acyl esters as carbon sources.

  20. Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs[S

    PubMed Central

    Li, Qingling; Zhang, Shenghui; Berthiaume, Jessica M.; Simons, Brigitte; Zhang, Guo-Fang

    2014-01-01

    A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80–114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs. PMID:24367045

  1. Chemoenzymatic Synthesis of Acyl Coenzyme A Substrates Enables in Situ Labeling of Small Molecules and Proteins.

    PubMed

    Agarwal, Vinayak; Diethelm, Stefan; Ray, Lauren; Garg, Neha; Awakawa, Takayoshi; Dorrestein, Pieter C; Moore, Bradley S

    2015-09-18

    A chemoenzymatic approach to generate fully functional acyl coenzyme A molecules that are then used as substrates to drive in situ acyl transfer reactions is described. Mass spectrometry based assays to verify the identity of acyl coenzyme A enzymatic products are also illustrated. The approach is responsive to a diverse array of carboxylic acids that can be elaborated to their corresponding coenzyme A thioesters, with potential applications in wide-ranging chemical biology studies that utilize acyl coenzyme A substrates.

  2. Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

    PubMed Central

    Duckworth, Benjamin P.; Nelson, Kathryn M.; Aldrich, Courtney C.

    2013-01-01

    Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases. PMID:22283817

  3. Establishment of Intestinal Bacteriology

    PubMed Central

    MITSUOKA, Tomotari

    2014-01-01

    Research on intestinal bacteria began around the end of the 19th century. During the last 5 decades of the 20th century, research on the intestinal microbiota made rapid progress. At first, in my work, I first developed a method of comprehensive analysis of the intestinal microbiota, and then I established classification and identification methods for intestinal anaerobes. Using these methods I discovered a number of ecological rules governing the intestinal microbiota and the role of the intestinl microbiota in health and disease. Moreover, using germfree animals, it was proven that the intestinal microbiota has a role in carcinogenesis and aging in the host. Thus, a new interdisciplinary field, “intestinal bacteriology” was established. PMID:25032084

  4. Characterization of a serine hydrolase targeted by acyl-protein thioesterase inhibitors in Toxoplasma gondii.

    PubMed

    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

    2013-09-20

    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

  5. 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

  6. Intestinal Disaccharidase Activity in Patients with Autism: Effect of Age, Gender, and Intestinal Inflammation

    ERIC Educational Resources Information Center

    Kushak, Rafail I.; Lauwers, Gregory Y.; Winter, Harland S.; Buie, Timothy M.

    2011-01-01

    Intestinal disaccharidase activities were measured in 199 individuals with autism to determine the frequency of enzyme deficiency. All patients had duodenal biopsies that were evaluated morphologically and assayed for lactase, sucrase, and maltase activity. Frequency of lactase deficiency was 58% in autistic children less than or equal to 5 years…

  7. Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase*

    PubMed Central

    Yao, Jiangwei; Dodson, V. Joshua; Frank, Matthew W.; Rock, Charles O.

    2015-01-01

    The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents. PMID:26195634

  8. Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase.

    PubMed

    Yao, Jiangwei; Dodson, V Joshua; Frank, Matthew W; Rock, Charles O

    2015-09-01

    The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents. PMID:26195634

  9. Acyl-ACP thioesterases from castor (Ricinus communis L.): an enzymatic system appropriate for high rates of oil synthesis and accumulation.

    PubMed

    Sánchez-García, Alicia; Moreno-Pérez, Antonio J; Muro-Pastor, Alicia M; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2010-06-01

    Acyl-acyl carrier protein (ACP) thioesterases are enzymes that terminate the intraplastidial fatty acid synthesis in plants by hydrolyzing the acyl-ACP intermediates and releasing free fatty acids to be incorporated into glycerolipids. These enzymes are classified in two families, FatA and FatB, which differ in amino acid sequence and substrate specificity. In the present work, both FatA and FatB thioesterases were cloned, sequenced and characterized from castor (Ricinus communis) seeds, a crop of high interest in oleochemistry. Single copies of FatA and FatB were found in castor resulting to be closely related with those of Jatropha curcas. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves. The contribution of these two enzymes to the synthesis of castor oil is discussed.

  10. Effect of age and diet composition on activity of pancreatic enzymes in birds.

    PubMed

    Brzęk, Paweł; Ciminari, M Eugenia; Kohl, Kevin D; Lessner, Krista; Karasov, William H; Caviedes-Vidal, Enrique

    2013-07-01

    Digestive enzymes produced by the pancreas and intestinal epithelium cooperate closely during food hydrolysis. Therefore, activities of pancreatic and intestinal enzymes processing the same substrate can be hypothesized to change together in unison, as well as to be adjusted to the concentration of their substrate in the diet. However, our knowledge of ontogenetic and diet-related changes in the digestive enzymes of birds is limited mainly to intestinal enzymes; it is largely unknown whether they are accompanied by changes in activities of enzymes produced by the pancreas. Here, we analyzed age- and diet-related changes in activities of pancreatic enzymes in five passerine and galloanserine species, and compared them with simultaneous changes in activities of intestinal enzymes. Mass-specific activity of pancreatic amylase increased with age in young house sparrows but not in zebra finches, in agreement with changes in typical dietary starch content and activity of intestinal maltase. However, we found little evidence for the presence of adaptive, diet-related modulation of pancreatic enzymes in both passerine and galloanserine species, even though in several cases the same birds adaptively modulated activities of their intestinal enzymes. In general, diet-related changes in mass-specific activities of pancreatic and intestinal enzymes were not correlated. We conclude that activity of pancreatic enzymes in birds is under strong genetic control, which enables evolutionary adjustment to typical diet composition but is less adept for short term, diet-related flexibility.

  11. 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

  12. Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates

    SciTech Connect

    Banerjee, R.V.; Shane, B.; McGuire, J.J.; Coward, J.K.

    1988-12-13

    The transfer of /sup 17/O and/or /sup 18/O from (COOH-/sup 17/O or -/sup 18/O) enriched substrates to inorganic phosphate (P/sub i/) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation. COOH-/sup 18/O-labeled folate, methotrexate, and dihydropteroate, in addition to (/sup 17/O)-glutamate, were synthesized and used as substrates for folylpolyglutamate synthetase (FPGS) isolated from Escherichia coli, hog liver, and rat liver and for dihydrofolate synthetase (DHFS) isolated from E. coli. P/sub i/ was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for /sup 17/O and /sup 18/O enrichment by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectroscopy. In the reactions catalyzed by the E. coli enzymes, both NMR and quantitative mass spectral analyses established that transfer of the oxygen isotope from the substrate /sup 18/O-enriched carboxyl group to P/sub i/ occurred, thereby providing strong evidence for an acyl phosphate intermediate in both the FPGS- and DHFS-catalyzed reactions. Similar oxygen-transfer experiments were carried out by use of two mammalian enzymes. The small amounts of P/sub i/ obtained from reactions catalyzed by these less abundant FPGS proteins precluded the use of NMR techniques. However, mass spectral analysis of the TMP derived from the mammalian FPGS-catalyzed reactions showed clearly that /sup 18/O transfer had occurred.

  13. Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane anchorage region

    SciTech Connect

    Chakravarty, R.; Rice, R.H.

    1989-01-05

    The membrane-bound form of keratinocyte transglutaminase was found to be labeled by addition of (/sup 3/H) acetic, (/sup 3/H)myristic, or (/sup 3/H)palmitic acids to the culture medium of human epidermal cells. Acid methanolysis and high performance liquid chromatography analysis of palmitate-labeled transglutaminase yielded only methyl palmitate. In contrast, analysis of the myristate-labeled protein yielded approximately 40% methyl myristate and 60% methyl palmitate. Incorporation of neither label was significantly affected by cycloheximide inhibition of protein synthesis. The importance of the fatty acid moiety for membrane anchorage was demonstrated in three ways. First, the enzyme was solubilized from the particulate fraction of cell extracts by treatment with neutral 1 M hydroxylamine, which was sufficient to release the fatty acid label. Second, solubilization of active enzyme from the particulate fraction upon mild trypsin treatment resulted in a reduction in size by approximately 10 kDa and removal of the fatty acid radiolabels. Third, the small fraction of soluble transglutaminase in cell extracts was found almost completely to lack fatty acid labeling. Keratinocyte transglutaminase translated from poly(A+) RNA in a reticulocyte cell-free system was indistinguishable in size from the native enzyme, suggesting anchorage requires only minor post-translational processing. Thus, the data are highly compatible with membrane anchorage by means of fatty acid acylation within 10 kDa of the NH/sub 2/ or COOH terminus.

  14. Mechanistic studies of malonic acid-mediated in situ acylation.

    PubMed

    Chandra, Koushik; Naoum, Johnny N; Roy, Tapta Kanchan; Gilon, Chaim; Gerber, R Benny; Friedler, Assaf

    2015-09-01

    We have previously introduced an easy to perform, cost-effective and highly efficient acetylation technique for solid phase synthesis (SPPS). Malonic acid is used as a precursor and the reaction proceeds via a reactive ketene that acetylates the target amine. Here we present a detailed mechanistic study of the malonic acid-mediated acylation. The influence of reaction conditions, peptide sequence and reagents was systematically studied. Our results show that the methodology can be successfully applied to different types of peptides and nonpeptidic molecules irrespective of their structure, sequence, or conformation. Using alkyl, phenyl, and benzyl malonic acid, we synthesized various acyl peptides with almost quantitative yields. The ketenes obtained from the different malonic acid derived precursors were characterized by in situ (1) H-NMR. The reaction proceeded in short reaction times and resulted in excellent yields when using uronium-based coupling agents, DIPEA as a base, DMF/DMSO/NMP as solvents, Rink amide/Wang/Merrifield resins, temperature of 20°C, pH 8-12 and 5 min preactivation at inert atmosphere. The reaction was unaffected by Lewis acids, transition metal ions, surfactants, or salt. DFT studies support the kinetically favorable concerted mechanism for CO2 and ketene formation that leads to the thermodynamically stable acylated products. We conclude that the malonic acid-mediated acylation is a general method applicable to various target molecules. PMID:25846609

  15. Acyl-CoA-Binding Proteins (ACBPs) in Plant Development.

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-01-01

    Acyl-CoA-binding proteins (ACBPs) play a pivotal role in fatty acid metabolism because they can transport medium- and long-chain acyl-CoA esters. In eukaryotic cells, ACBPs are involved in intracellular trafficking of acyl-CoA esters and formation of a cytosolic acyl-CoA pool. In addition to these ubiquitous functions, more specific non-redundant roles of plant ACBP subclasses are implicated by the existence of multigene families with variable molecular masses, ligand specificities, functional domains (e.g. protein-protein interaction domains), subcellular locations and gene expression patterns. In this chapter, recent progress in the characterization of ACBPs from the model dicot plant, Arabidopsis thaliana, and the model monocot, Oryza sativa, and their emerging roles in plant growth and development are discussed. The functional significance of respective members of the plant ACBP families in various developmental and physiological processes such as seed development and germination, stem cuticle formation, pollen development, leaf senescence, peroxisomal fatty acid β-oxidation and phloem-mediated lipid transport is highlighted.

  16. Preservation of polyunsaturated fatty acyl glycerides via intramolecular antioxidant coupling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ferulic acid and its esters are known to be effective antioxidants. Feruloyl di-gamma-linolenoylglycerol was assessed for its ability to serve as an antioxidant for preventing the oxidation of its gamma-linolenoyl polyunsaturated fatty acyl groups in model membrane phospholipid vesicles. The molec...

  17. Lubricity characteristics of seed oils modified by acylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chemically modified seed oils via acylation of epoxidized and polyhydroxylated derivatives were investigated for their potential as candidates for lubrication. The native oil was preliminarily epoxidized and ring-opened in a one-pot reaction using formic acid-H2O2 followed by aqueous HCl treatment t...

  18. Acyl-CoA-Binding Proteins (ACBPs) in Plant Development.

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-01-01

    Acyl-CoA-binding proteins (ACBPs) play a pivotal role in fatty acid metabolism because they can transport medium- and long-chain acyl-CoA esters. In eukaryotic cells, ACBPs are involved in intracellular trafficking of acyl-CoA esters and formation of a cytosolic acyl-CoA pool. In addition to these ubiquitous functions, more specific non-redundant roles of plant ACBP subclasses are implicated by the existence of multigene families with variable molecular masses, ligand specificities, functional domains (e.g. protein-protein interaction domains), subcellular locations and gene expression patterns. In this chapter, recent progress in the characterization of ACBPs from the model dicot plant, Arabidopsis thaliana, and the model monocot, Oryza sativa, and their emerging roles in plant growth and development are discussed. The functional significance of respective members of the plant ACBP families in various developmental and physiological processes such as seed development and germination, stem cuticle formation, pollen development, leaf senescence, peroxisomal fatty acid β-oxidation and phloem-mediated lipid transport is highlighted. PMID:27023243

  19. Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2-lysophospholipids in biological samples by LC-MS/MS

    PubMed Central

    Okudaira, Michiyo; Inoue, Asuka; Shuto, Akira; Nakanaga, Keita; Kano, Kuniyuki; Makide, Kumiko; Saigusa, Daisuke; Tomioka, Yoshihisa; Aoki, Junken

    2014-01-01

    Lysophospholipids (LysoGPs) serve as lipid mediators and precursors for synthesis of diacyl phospholipids (GPs). LysoGPs detected in cells have various acyl chains attached at either the sn-1 or sn-2 position of the glycerol backbone. In general, acyl chains at the sn-2 position of 2-acyl-1-LysoGPs readily move to the sn-1 position, generating 1-acyl-2-lyso isomers by a nonenzymatic reaction called intra-molecular acyl migration, which has hampered the detection of 2-acyl-1-LysoGPs in biological samples. In this study, we developed a simple and versatile method to separate and quantify 2-acyl-1- and 1-acyl-2-LysoGPs. The main point of the method was to extract LysoGPs at pH 4 and 4°C, conditions that were found to completely eliminate the intra-molecular acyl migration. Under the present conditions, the relative amounts of 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs did not change at least for 1 week. Further, in LysoGPs extracted from cells and tissues under the present conditions, most of the saturated fatty acids (16:0 and 18:0) were found in the sn-1 position of LysoGPs, while most of the PUFAs (18:2, 20:4, 22:6) were found in the sn-2 position. Thus the method can be used to elucidate the in vivo role of 2-acyl-1-LysoGPs. PMID:25114169

  20. Enzyme action in the regulation of plant hormone responses.

    PubMed

    Westfall, Corey S; Muehler, Ashley M; Jez, Joseph M

    2013-07-01

    Plants synthesize a chemically diverse range of hormones that regulate growth, development, and responses to environmental stresses. The major classes of plant hormones are specialized metabolites with exquisitely tailored perception and signaling systems, but equally important are the enzymes that control the dose and exposure to the bioactive forms of these molecules. Here, we review new insights into the role of enzyme families, including the SABATH methyltransferases, the methylesterases, the GH3 acyl acid-amido synthetases, and the hormone peptidyl hydrolases, in controlling the biosynthesis and modifications of plant hormones and how these enzymes contribute to the network of chemical signals responsible for plant growth, development, and environmental adaptation.

  1. Draft Genome Sequence of the Thermophile Thermus filiformis ATCC 43280, Producer of Carotenoid-(Di)glucoside-Branched Fatty Acid (Di)esters and Source of Hyperthermostable Enzymes of Biotechnological Interest

    PubMed Central

    Mandelli, Fernanda; Oliveira Ramires, Brenda; Couger, Matthew Brian; Paixão, Douglas A. A.; Camilo, Cesar M.; Polikarpov, Igor; Prade, Rolf

    2015-01-01

    Here, we present the draft genome sequence of Thermus filiformis strain ATCC 43280, a thermophile bacterium capable of producing glycosylated carotenoids acylated with branched fatty acids and enzymes of biotechnological potential. PMID:25977443

  2. 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

  3. Thermodynamically based solvent design for enzymatic saccharide acylation with hydroxycinnamic acids in non-conventional media.

    PubMed

    Zeuner, Birgitte; Kontogeorgis, Georgios M; Riisager, Anders; Meyer, Anne S

    2012-02-15

    Enzyme-catalyzed synthesis has been widely studied with lipases (EC 3.1.1.3), but feruloyl esterases (FAEs; EC 3.1.1.73) may provide advantages such as higher substrate affinity and regioselectivity in the synthesis of hydroxycinnamate saccharide esters. These compounds are interesting because of their amphiphilicity and antioxidative potential. Synthetic reactions using mono- or disaccharides as one of the substrates may moreover direct new routes for biomass upgrading in the biorefinery. The paper reviews the available data for enzymatic hydroxycinnamate saccharide ester synthesis in organic solvent systems as well as other enzymatic hydroxycinnamate acylations in ionic liquid systems. The choice of solvent system is highly decisive for enzyme stability, selectivity, and reaction yields in these synthesis reactions. To increase the understanding of the reaction environment and to facilitate solvent screening as a crucial part of the reaction design, the review explores the use of activity coefficient models for describing these systems and - more importantly - the use of group contribution model UNIFAC and quantum chemistry based COSMO-RS for thermodynamic predictions and preliminary solvent screening. Surfactant-free microemulsions of a hydrocarbon, a polar alcohol, and water are interesting solvent systems because they accommodate different substrate and product solubilities and maintain enzyme stability. Ionic liquids may provide advantages as solvents in terms of increased substrate and product solubility, higher reactivity and selectivity, as well as tunable physicochemical properties, but their design should be carefully considered in relation to enzyme stability. The treatise shows that thermodynamic modeling tools for solvent design provide a new toolbox to design enzyme-catalyzed synthetic reactions from biomass sources. PMID:22154740

  4. Enhanced Enzymatic Preparation of Biodiesel Using Ricinoleic Acid as Acyl Donor: Optimization Using Response Surface Methodology.

    PubMed

    Wang, Ping; Sun, Shangde

    2016-09-01

    Castor oil methyl ester is a kind of biodiesel from castor oil. However, in those previous methods for biodiesel preparation using castor oil as feedstock, glycerol was the main by-product, which had a strong blocking effect on the immobilized enzyme activity and affected the mass transfer of reaction system. For avoiding the negative effect of glycerol on the enzymatic esterification, biodiesel was prepared using ricinoleic acid (RA) as acyl donor. Enzyme screening was also studied, and the effects of reaction temperature, molar ratio of ricinoleic acid and methanol, enzyme load, and reaction time, on the preparation of castor methyl ester were also evaluated. Response surface methodology (RSM) was used to optimize the interaction effect of reaction variables (reaction temperature (30-70°C), enzyme load (2-7%; relative to the weight of total substrates), molar ratio of methanol to ricinoleic acid (2:1-10:1), and reaction time (0.5-2.5 h)) on the acid value (AV) and the degree of esterification (DE). Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of AV and DE. The optimum preparation conditions were as follows: reaction temperature, 48.2°C; enzyme load, 5.8%; molar ratio of methanol to ricinoleic acid, 5.56:1; reaction time, 2.36 h. Under these conditions, the AV and DE of the esterification reaction are 10.36±1.05 mgKOH/g and 94.03±0.60%, respectively. The relationship between initial reaction rate and temperature was also established, and the activation energy (Ea) of the enzymatic esterification is 33.87 KJ/mol. PMID:27477073

  5. Bacterial acyl-CoA mutase specifically catalyzes coenzyme B12-dependent isomerization of 2-hydroxyisobutyryl-CoA and (S)-3-hydroxybutyryl-CoA.

    PubMed

    Yaneva, Nadya; Schuster, Judith; Schäfer, Franziska; Lede, Vera; Przybylski, Denise; Paproth, Torsten; Harms, Hauke; Müller, Roland H; Rohwerder, Thore

    2012-05-01

    Coenzyme B(12)-dependent acyl-CoA mutases are radical enzymes catalyzing reversible carbon skeleton rearrangements in carboxylic acids. Here, we describe 2-hydroxyisobutyryl-CoA mutase (HCM) found in the bacterium Aquincola tertiaricarbonis as a novel member of the mutase family. HCM specifically catalyzes the interconversion of 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl-CoA. Like isobutyryl-CoA mutase, HCM consists of a large substrate- and a small B(12)-binding subunit, HcmA and HcmB, respectively. However, it is thus far the only acyl-CoA mutase showing substrate specificity for hydroxylated carboxylic acids. Complete loss of 2-hydroxyisobutyric acid degradation capacity in hcmA and hcmB knock-out mutants established the central role of HCM in A. tertiaricarbonis for degrading substrates bearing a tert-butyl moiety, such as the fuel oxygenate methyl tert-butyl ether (MTBE) and its metabolites. Sequence analysis revealed several HCM-like enzymes in other bacterial strains not related to MTBE degradation, indicating that HCM may also be involved in other pathways. In all strains, hcmA and hcmB are associated with genes encoding for a putative acyl-CoA synthetase and a MeaB-like chaperone. Activity and substrate specificity of wild-type enzyme and active site mutants HcmA I90V, I90F, and I90Y clearly demonstrated that HCM belongs to a new subfamily of B(12)-dependent acyl-CoA mutases. PMID:22433853

  6. Clarification of the Mechanism of Acylation Reaction and Origin of Substrate Specificity of the Serine-Carboxyl Peptidase Sedolisin through QM/MM Free Energy Simulations

    SciTech Connect

    Xu, Qin; Yao, Jianzhuang; Wiodawer, Alexander; Guo, Hong

    2011-01-01

    Quantum mechanical/molecular mechanical (QM/MM) free energy simulations are applied for understanding the mechanism of the acylation reaction catalyzed by sedolisin, a representative serine-carboxyl peptidase, leading to the acyl-enzyme (AE) and first product from the enzyme-catalyzed reaction. One of the interesting questions to be addressed in this work is the origin of the substrate specificity of sedolisin that shows a relatively high activity on the substrates with Glu at P1 site. It is shown that the bond making and breaking events of the acylation reaction involving a peptide substrate (LLE*FL) seem to be accompanied by local conformational changes, proton transfers as well as the formation of alternative hydrogen bonds. The results of the simulations indicate that the conformational change of Glu at P1 site and its formation of a low barrier hydrogen bond with Asp-170 (along with the transient proton transfer) during the acylation reaction might play a role in the relatively high specificity for the substrate with Glu at P1 site. The role of some key residues in the catalysis is confirmed through free energy simulations. Glu-80 is found to act as a general base to accept a proton from Ser-287 during the nucleophilic attack and then as a general acid to protonate the leaving group (N H of P1 -Phe) during the cleavage of the scissile peptide bond. Another acidic residue, Asp-170, acts as a general acid catalyst to protonate the carbonyl of P1-Glu during the formation of the tetrahedral intermediate and as a general base for the formation of the acyl-enzyme. The energetic results from the free energy simulations support the importance of proton transfer from Asp-170 to the carbonyl of P1-Glu in the stabilization of the tetrahedral intermediate and the formation of a low-barrier hydrogen bond between the carboxyl group of P1-Glu and Asp-170 in the lowering of the free energy barrier for the cleavage of the peptide bond. Detailed analyses of the proton transfers

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

    PubMed

    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.

  8. Dyslipidaemia--hepatic and intestinal cross-talk.

    PubMed

    Tomkin, Gerald H

    2010-06-01

    Cholesterol metabolism is tightly regulated with the majority of de novo cholesterol synthesis occurring in the liver and intestine. 3 Hydroxy-3-methylglutaryl coenzyme A reductase, a major enzyme involved in cholesterol synthesis, is raised in both liver and intestine in diabetic animals. Niemann PickC1-like1 protein regulates cholesterol absorption in the intestine and facilitates cholesterol transport through the liver. There is evidence to suggest that the effect of inhibition of Niemann PickC1-like1 lowers cholesterol through its effect not only in the intestine but also in the liver. ATP binding cassette proteins G5/G8 regulate cholesterol re-excretion in the intestine and in the liver, cholesterol excretion into the bile. Diabetes is associated with reduced ATP binding cassette protein G5/G8 expression in both the liver and intestine in animal models. Microsomal triglyceride transfer protein is central to the formation of the chylomicron in the intestine and VLDL in the liver. Microsomal triglyceride transfer protein mRNA is increased in diabetes in both the intestine and liver. Cross-talk between the intestine and liver is poorly documented in humans due to the difficulty in obtaining liver biopsies but animal studies are fairly consistent in showing relationships that explain in part mechanisms involved in cholesterol homeostasis.

  9. A novel and widespread class of ketosynthase is responsible for the head-to-head condensation of two acyl moieties in bacterial pyrone biosynthesis

    PubMed Central

    Kresovic, Darko; Schempp, Florence; Cheikh-Ali, Zakaria

    2015-01-01

    Summary The biosynthesis of photopyrones, novel quorum sensing signals in Photorhabdus, has been studied by heterologous expression of the photopyrone synthase PpyS catalyzing the head-to-head condensation of two acyl moieties. The biochemical mechanism of pyrone formation has been investigated by amino acid exchange and bioinformatic analysis. Additionally, the evolutionary origin of PpyS has been studied by phylogenetic analyses also revealing homologous enzymes in Pseudomonas sp. GM30 responsible for the biosynthesis of pseudopyronines including a novel derivative. Moreover this novel class of ketosynthases is only distantly related to other pyrone-forming enzymes identified in the biosynthesis of the potent antibiotics myxopyronin and corallopyronin. PMID:26425196

  10. Direct N-acylation of azoles via a metal-free catalyzed oxidative cross-coupling strategy.

    PubMed

    Zhao, Jingjing; Li, Pan; Xia, Chungu; Li, Fuwei

    2014-05-11

    The KI-catalyzed N-acylation of azoles via direct oxidative coupling of C-H and N-H bonds has been developed. It could be smoothly scaled up to gram synthesis of acyl azoles. The reaction occurred by the coupling of acyl radicals and azoles to form the acyl azole radical anion, followed by its further oxidation.

  11. Intestinal lymphangiectasia in children

    PubMed Central

    Isa, Hasan M.; Al-Arayedh, Ghadeer G.; Mohamed, Afaf M.

    2016-01-01

    Intestinal lymphangiectasia (IL) is a rare disease characterized by dilatation of intestinal lymphatics. It can be classified as primary or secondary according to the underlying etiology. The clinical presentations of IL are pitting edema, chylous ascites, pleural effusion, acute appendicitis, diarrhea, lymphocytopenia, malabsorption, and intestinal obstruction. The diagnosis is made by intestinal endoscopy and biopsies. Dietary modification is the mainstay in the management of IL with a variable response. Here we report 2 patients with IL in Bahrain who showed positive response to dietary modification. PMID:26837404

  12. Intestinal transplantation: a review.

    PubMed

    Desai, Chirag Sureshchandra; Khan, Khalid Mahmood; Girlanda, Raffaele; Fishbein, Thomas M

    2012-09-01

    Parenteral nutrition is a life-saving therapy for patients with intestinal failure. Intestinal transplantation is now recognized as a treatment for patients who develop complications of parenteral nutrition and in whom attempts at intestinal rehabilitation have failed. Patients with parenteral nutrition related liver disease will require a liver graft typically part of a multivisceral transplant. Isolated intestinal transplants are more commonly performed in adults while multivisceral transplants are most commonly performed in infants. Isolated intestinal transplants have the best short-term outcome, with over 80 % survival at 1 year. Patients requiring multivisceral transplants have a high rate of attrition with a 1 year survival less than 70 %. Prognostic factors for a poor outcome include patient hospitalization at the time of transplant and donor age greater than 40 years while systemic sepsis and acute rejection are the major determinant of early postoperative outcome. For patients surviving the first year the outcome of transplantation of the liver in addition to intestine affords some survival advantage though long-term outcome does not yet match other abdominal organs. Outcomes for intestinal retransplantation are poor as a result of immunology and patient debility. Overall intestinal transplantation continues to develop and is a clear indication with cost and quality of life advantages in patients with intestinal failure that do not remain stable on parenteral nutrition.

  13. Production of a Brassica napus low-molecular mass acyl-coenzyme A-binding protein in Arabidopsis alters the acyl-coenzyme A pool and acyl composition of oil in seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expressio...

  14. Tacrolimus-Induced Intestinal Angioedema: Diagnosis by Capsule Endoscopy

    PubMed Central

    Zvidi, I.; Gal, E.; Rachamimov, R.; Niv, Y.

    2007-01-01

    Small intestinal angioedema has been reported with angiotensin converting enzyme inhibitors therapy, but not in implanted patients treated with tacrolimus. We present a kidney transplanted patient, hospitalized with severe diarrhea, diagnosed with tacrolimus-induced intestinal angioedema with abdominal computerized tomography and capsule endoscopy. To the best of our knowledge this is the first described case of tacrolimus-induced small bowel angioedema diagnosed with capsule endoscopy. PMID:20376210

  15. 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

    2016-01-01

    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

  16. 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.

  17. A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.

    PubMed

    Garver, W S; Kemp, J D; Kuehn, G D

    1992-12-01

    Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.

  18. Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA

    PubMed Central

    Albesa-Jové, David; Svetlíková, Zuzana; Tersa, Montse; Sancho-Vaello, Enea; Carreras-González, Ana; Bonnet, Pascal; Arrasate, Pedro; Eguskiza, Ander; Angala, Shiva K.; Cifuente, Javier O.; Korduláková, Jana; Jackson, Mary; Mikušová, Katarína; Guerin, Marcelo E.

    2016-01-01

    The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl–CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl–CoA analog. The structures reveal an α/β architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design. PMID:26965057

  19. Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase

    PubMed Central

    Lanyon-Hogg, Thomas; Masumoto, Naoko; Bodakh, George; Konitsiotis, Antonio D.; Thinon, Emmanuelle; Rodgers, Ursula R.; Owens, Raymond J.; Magee, Anthony I.; Tate, Edward W.

    2016-01-01

    In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed “RU-SKI”) class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a), RU-SKI 43 (9b), RU-SKI 101 (9c), and RU-SKI 201 (9d) were profiled for activity in the related article “Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase” (Lanyon-Hogg et al., 2015) [1]. 1H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors. PMID:27077078

  20. 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

  1. Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase.

    PubMed

    Lanyon-Hogg, Thomas; Masumoto, Naoko; Bodakh, George; Konitsiotis, Antonio D; Thinon, Emmanuelle; Rodgers, Ursula R; Owens, Raymond J; Magee, Anthony I; Tate, Edward W

    2016-06-01

    In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed "RU-SKI") class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a), RU-SKI 43 (9b), RU-SKI 101 (9c), and RU-SKI 201 (9d) were profiled for activity in the related article "Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase" (Lanyon-Hogg et al., 2015) [1]. (1)H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors. PMID:27077078

  2. Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA.

    PubMed

    Albesa-Jové, David; Svetlíková, Zuzana; Tersa, Montse; Sancho-Vaello, Enea; Carreras-González, Ana; Bonnet, Pascal; Arrasate, Pedro; Eguskiza, Ander; Angala, Shiva K; Cifuente, Javier O; Korduláková, Jana; Jackson, Mary; Mikušová, Katarína; Guerin, Marcelo E

    2016-01-01

    The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl-CoA analog. The structures reveal an α/β architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design. PMID:26965057

  3. The enzymes of bacterial census and censorship.

    PubMed

    Fast, Walter; Tipton, Peter A

    2012-01-01

    N-Acyl-L-homoserine lactones (AHLs) are a major class of quorum-sensing signals used by Gram-negative bacteria to regulate gene expression in a population-dependent manner, thereby enabling group behavior. Enzymes capable of generating and catabolizing AHL signals are of significant interest for the study of microbial ecology and quorum-sensing pathways, for understanding the systems that bacteria have evolved to interact with small-molecule signals, and for their possible use in therapeutic and industrial applications. The recent structural and functional studies reviewed here provide a detailed insight into the chemistry and enzymology of bacterial communication.

  4. The enzymes of bacterial census and censorship.

    PubMed

    Fast, Walter; Tipton, Peter A

    2012-01-01

    N-Acyl-L-homoserine lactones (AHLs) are a major class of quorum-sensing signals used by Gram-negative bacteria to regulate gene expression in a population-dependent manner, thereby enabling group behavior. Enzymes capable of generating and catabolizing AHL signals are of significant interest for the study of microbial ecology and quorum-sensing pathways, for understanding the systems that bacteria have evolved to interact with small-molecule signals, and for their possible use in therapeutic and industrial applications. The recent structural and functional studies reviewed here provide a detailed insight into the chemistry and enzymology of bacterial communication. PMID:22099187

  5. Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects

    PubMed Central

    Wang, Yan; Song, Qinghao; Zhang, Xiao-Hua

    2016-01-01

    Marine microorganisms produce a series of promising enzymes that have been widely used or are potentially valuable for our daily life. Both classic and newly developed biochemistry technologies have been broadly used to study marine and terrestrial microbiological enzymes. In this brief review, we provide a research update and prospects regarding regulatory mechanisms and related strategies of acyl-homoserine lactones (AHL) lactonase, which is an important but largely unexplored enzyme. We also detail the status and catalytic mechanism of the main types of polysaccharide-degrading enzymes that broadly exist among marine microorganisms but have been poorly explored. In order to facilitate understanding, the regulatory and synthetic biology strategies of terrestrial microorganisms are also mentioned in comparison. We anticipate that this review will provide an outline of multiple strategies for promising marine microbial enzymes and open new avenues for the exploration, engineering and application of various enzymes. PMID:27669268

  6. Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects.

    PubMed

    Wang, Yan; Song, Qinghao; Zhang, Xiao-Hua

    2016-09-22

    Marine microorganisms produce a series of promising enzymes that have been widely used or are potentially valuable for our daily life. Both classic and newly developed biochemistry technologies have been broadly used to study marine and terrestrial microbiological enzymes. In this brief review, we provide a research update and prospects regarding regulatory mechanisms and related strategies of acyl-homoserine lactones (AHL) lactonase, which is an important but largely unexplored enzyme. We also detail the status and catalytic mechanism of the main types of polysaccharide-degrading enzymes that broadly exist among marine microorganisms but have been poorly explored. In order to facilitate understanding, the regulatory and synthetic biology strategies of terrestrial microorganisms are also mentioned in comparison. We anticipate that this review will provide an outline of multiple strategies for promising marine microbial enzymes and open new avenues for the exploration, engineering and application of various enzymes.

  7. Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects.

    PubMed

    Wang, Yan; Song, Qinghao; Zhang, Xiao-Hua

    2016-01-01

    Marine microorganisms produce a series of promising enzymes that have been widely used or are potentially valuable for our daily life. Both classic and newly developed biochemistry technologies have been broadly used to study marine and terrestrial microbiological enzymes. In this brief review, we provide a research update and prospects regarding regulatory mechanisms and related strategies of acyl-homoserine lactones (AHL) lactonase, which is an important but largely unexplored enzyme. We also detail the status and catalytic mechanism of the main types of polysaccharide-degrading enzymes that broadly exist among marine microorganisms but have been poorly explored. In order to facilitate understanding, the regulatory and synthetic biology strategies of terrestrial microorganisms are also mentioned in comparison. We anticipate that this review will provide an outline of multiple strategies for promising marine microbial enzymes and open new avenues for the exploration, engineering and application of various enzymes. PMID:27669268

  8. Production of N-acyl Homoserine Lactones and Virulence Factors of Waterborne Aeromonas hydrophila.

    PubMed

    Chu, Weihua; Liu, Yongwang; Jiang, Yan; Zhu, Wei; Zhuang, Xiyi

    2013-09-01

    Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. The purpose of this study was to investigate the production of N-acyl-homoserine lactone (AHL) signal molecules and some virulence factors, including hemolysins, proteases, extracellular nucleases production and cytotoxicity by waterborne Aeromonas hydrophila. A total of 24 strains isolated from fresh-water or diseased fish were used in the study. The majority A.hydrophila strains produce two AHL molecules (21/24), one is N-butanoyl homoserine lactone (BHL), and the other is N-hexanoyl homoserine lactone (HHL) according to thin-layer chromatography analysis. Among the virulence factors tested, more than 83 % of the isolates produced β haemolysin when inoculated on sheep blood agar, only 50 % of the isolates displayed DNase activity, 75 % of the isolates shown proteolytic activity on skimmed milk plate, and cytotoxic activity was detected in 20 of 24 of the isolates. The strains producing AHLs possessed one or more virulence factors. In conclusion, the production of quorum sensing signal molecules is common among the strains that we examined, and there seems to some relationships between quorum sensing signal production and virulence factors in A. hydrophila.

  9. A negative regulating element controlling transcription of the gene encoding acyl-CoA oxidase in Saccharomyces cerevisiae.

    PubMed Central

    Wang, T W; Lewin, A S; Small, G M

    1992-01-01

    Peroxisomes are induced in Saccharomyces cerevisiae when this yeast is grown in the presence of oleate, and are repressed when glucose is supplied as the carbon source. Concomitant with this is an induction/repression of peroxisomal beta-oxidation enzymes. We are investigating the transcriptional control of acyl-CoA oxidase, the first and rate-limiting enzyme in the peroxisomal beta-oxidation cycle. The promoter region of POX1 from S. cerevisiae has been analyzed in POX1/lacZ fusions. Expression of the POX1/lacZ fusion protein underwent glucose repression and oleate induction. By deletion, DNA band shift and DNase I footprinting analyses we have identified a region that is involved in transcriptional repression of POX1. Elimination of this DNA sequence results in constitutive expression of POX1 when S. cerevisiae is grown on a fermentable carbon source or glycerol. Images PMID:1630920

  10. Dynamics of the Heat Stress Response of Ceramides with Different Fatty-Acyl Chain Lengths in Baker's Yeast.

    PubMed

    Chen, Po-Wei; Fonseca, Luis L; Hannun, Yusuf A; Voit, Eberhard O

    2015-08-01

    The article demonstrates that computational modeling has the capacity to convert metabolic snapshots, taken sequentially over time, into a description of cellular, dynamic strategies. The specific application is a detailed analysis of a set of actions with which Saccharomyces cerevisiae responds to heat stress. Using time dependent metabolic concentration data, we use a combination of mathematical modeling, reverse engineering, and optimization to infer dynamic changes in enzyme activities within the sphingolipid pathway. The details of the sphingolipid responses to heat stress are important, because they guide some of the longer-term alterations in gene expression, with which the cells adapt to the increased temperature. The analysis indicates that all enzyme activities in the system are affected and that the shapes of the time trends in activities depend on the fatty-acyl CoA chain lengths of the different ceramide species in the system.

  11. Dynamics of the Heat Stress Response of Ceramides with Different Fatty-Acyl Chain Lengths in Baker's Yeast.

    PubMed

    Chen, Po-Wei; Fonseca, Luis L; Hannun, Yusuf A; Voit, Eberhard O

    2015-08-01

    The article demonstrates that computational modeling has the capacity to convert metabolic snapshots, taken sequentially over time, into a description of cellular, dynamic strategies. The specific application is a detailed analysis of a set of actions with which Saccharomyces cerevisiae responds to heat stress. Using time dependent metabolic concentration data, we use a combination of mathematical modeling, reverse engineering, and optimization to infer dynamic changes in enzyme activities within the sphingolipid pathway. The details of the sphingolipid responses to heat stress are important, because they guide some of the longer-term alterations in gene expression, with which the cells adapt to the increased temperature. The analysis indicates that all enzyme activities in the system are affected and that the shapes of the time trends in activities depend on the fatty-acyl CoA chain lengths of the different ceramide species in the system. PMID:26241868

  12. In vitro metabolism of canagliflozin in human liver, kidney, intestine microsomes, and recombinant uridine diphosphate glucuronosyltransferases (UGT) and the effect of genetic variability of UGT enzymes on the pharmacokinetics of canagliflozin in humans.

    PubMed

    Francke, Stephan; Mamidi, Rao N V S; Solanki, Bhavna; Scheers, Ellen; Jadwin, Andrew; Favis, Reyna; Devineni, Damayanthi

    2015-09-01

    O-glucuronidation is the major metabolic elimination pathway for canagliflozin. The objective was to identify enzymes and tissues involved in the formation of 2 major glucuronidated metabolites (M7 and M5) of canagliflozin and subsequently to assess the impact of genetic variations in these uridine diphosphate glucuronosyltransferases (UGTs) on in vivo pharmacokinetics in humans. In vitro incubations with recombinant UGTs revealed involvement of UGT1A9 and UGT2B4 in the formation of M7 and M5, respectively. Although M7 and M5 were formed in liver microsomes, only M7 was formed in kidney microsomes. Participants from 7 phase 1 studies were pooled for pharmacogenomic analyses. A total of 134 participants (mean age, 41 years; men, 63%; white, 84%) were included in the analysis. In UGT1A9*3 carriers, exposure of plasma canagliflozin (Cmax,ss , 11%; AUCτ,ss , 45%) increased relative to the wild type. An increase in exposure of plasma canagliflozin (Cmax,ss , 21%; AUCt,ss , 18%) was observed in participants with UGT2B4*2 genotype compared with UGT2B4*2 noncarriers. Metabolites further delineate the role of both enzymes. The pharmacokinetic findings in participants carrying the UGT1A9*3 and UGT2B4*2 allele implicate that UGT1A9 and UGT2B4 are involved in the metabolism of canagliflozin to M7 and M5, respectively. PMID:25827774

  13. The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids.

    PubMed

    Maeda, Isamu; Delessert, Syndie; Hasegawa, Seiko; Seto, Yoshiaki; Zuber, Sophie; Poirier, Yves

    2006-04-28

    The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.

  14. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis

    PubMed Central

    Nanson, Jeffrey D.; Himiari, Zainab; Swarbrick, Crystall M. D.; Forwood, Jade K.

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

  15. Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate

    PubMed Central

    Kormanik, Kaitlyn; Kang, Heejung; Cuebas, Dean; Vockley, Jerry; Mohsen, Al-Walid

    2012-01-01

    Sodium phenylbutyrate is used for treating urea cycle disorders, providing an alternative for ammonia excretion. Following conversion to its CoA ester, phenylbutyryl-CoA is postulated to undergo one round of β-oxidation to phenylacetyl-CoA, the active metabolite. Molecular modeling suggests that medium chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3), a key enzyme in straight chain fatty acid β-oxidation, could utilize phenylbutyryl-CoA as substrate. Moreover, phenylpropionyl-CoA has been shown to be a substrate for MCAD and its intermediates accumulate in patients with MCAD deficiency. We have examined the involvement of MCAD and other acyl-CoA dehydrogenases (ACADs) in the metabolism of phenylbutyryl-CoA. Anaerobic titration of purified recombinant human MCAD with phenylbutyryl-CoA caused changes in the MCAD spectrum that are similar to those induced by octanoyl-CoA, its bona fide substrate, and unique to the development of the charge transfer ternary complex. The calculated apparent dissociation constant (KD app) for these substrates was 2.16 μM and 0.12 μM, respectively. The MCAD reductive and oxidative half reactions were monitored using the electron transfer flavoprotein (ETF) fluorescence reduction assay. The catalytic efficiency and the Km for phenylbutyryl-CoA were 0.2 mM−1· sec−1 and 5.3 μM compared to 4.0 mM−1· sec−1 and 2.8 μM for octanoyl-CoA. Extracts of wild type and MCAD-deficient lymphoblast cells were tested for the ability to reduce ETF using phenylbutyryl-CoA as substrate. While ETF reduction activity was detected in extracts of wild type cells, it was undetectable in extracts of cells deficient in MCAD. The results are consistent with MCAD playing a key role in phenylbutyrate metabolism. PMID:23141465

  16. New acylated anthocyanins from purple yam and their antioxidant activity.

    PubMed

    Moriya, Chiemi; Hosoya, Takahiro; Agawa, Sayuri; Sugiyama, Yasumasa; Kozone, Ikuko; Shin-Ya, Kazuo; Terahara, Norihiko; Kumazawa, Shigenori

    2015-01-01

    Purple yam (Dioscorea alata L.), which is widely distributed in tropical and subtropical regions, is characterized by its color and viscosity. Previous studies have shown that purple yams contain a variety of acylated anthocyanins that exhibit higher levels of antioxidant activity than the corresponding nonacylated compounds. In this study, the pigments found in purple yams from the Philippines (D. alata) were isolated and evaluated in terms of antioxidant activity. Four new acylated anthocyanins, alanins (1-4) were isolated from the MeOH extracts of purple yam, which were subsequently determined to be cyanidin (1, 2, and 4) and peonidin (3) type compounds, along with four known anthocyanins (5-8). The structures of 1-4 were determined by spectroscopic methods, including NMR and MS analyses. The antioxidant activities of anthocyanins 1-8 were investigated using oxygen radical absorbing capacity and ferric reducing antioxidant power assays. PMID:25848974

  17. Reaction of Acylated Homoserine Lactone Bacterial Signaling Molecules with Oxidized Halogen Antimicrobials

    PubMed Central

    Borchardt, S. A.; Allain, E. J.; Michels, J. J.; Stearns, G. W.; Kelly, R. F.; McCoy, W. F.

    2001-01-01

    Oxidized halogen antimicrobials, such as hypochlorous and hypobromous acids, have been used extensively for microbial control in industrial systems. Recent discoveries have shown that acylated homoserine lactone cell-to-cell signaling molecules are important for biofilm formation in Pseudomonas aeruginosa, suggesting that biofouling can be controlled by interfering with bacterial cell-to-cell communication. This study was conducted to investigate the potential for oxidized halogens to react with acylated homoserine lactone-based signaling molecules. Acylated homoserine lactones containing a 3-oxo group were found to rapidly react with oxidized halogens, while acylated homoserine lactones lacking the 3-oxo functionality did not react. The Chromobacterium violaceum CV026 bioassay was used to determine the effects of such reactions on acylated homoserine lactone activity. The results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity. Experiments with the marine alga Laminaria digitata demonstrated that natural haloperoxidase systems are capable of mediating the deactivation of acylated homoserine lactones. This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga. The Chromobacterium violaceum activity assay illustrates that reactions between 3-oxo acylated homoserine lactone molecules and oxidized halogens do occur despite the presence of biofilm components at much greater concentrations. This work suggests that oxidized halogens may control biofilm not only via a cidal mechanism, but also by possibly interfering with 3-oxo acylated homoserine lactone-based cell signaling. PMID:11425738

  18. 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

  19. 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.

  20. Effects of long-chain fatty-acyl esters of coenzyme A and carnitine on cell-free rat heart preparations.

    PubMed

    Varela, A; Savino, E A

    1987-06-01

    The purpose of this study was to investigate the effects of fatty acyl CoA and carnitine esters on the glycolytic system of the rat heart. Using a respiring incubation mixture containing a whole-heart homogenate it was observed that oleoyl-CoA slowed down the glucose disappearance whereas lactate accumulation did not change. Experiments were also performed by means of an incubation mixture prepared with a soluble heart extract, considered to contain all glycolytic enzymes present in heart fibres. Palmitoyl-CoA or oleoyl-CoA as well as palmitoyl carnitine, added separately or together, were unable to alter the glucose disappearance and lactate accumulation in this mixture. These data suggest that long chain acyl-esters have not direct inhibitory actions on the heart glycolytic activity. However, CoA esters seem to exert indirect inhibitory effects which may be relevant to the myocardium under oxygen restriction situations.

  1. Six new acylated anthocyanins from red radish (Raphanus sativus).

    PubMed

    Tamura, Satoru; Tsuji, Kouji; Yongzhen, Piao; Ohnishi-Kameyama, Mayumi; Murakami, Nobutoshi

    2010-09-01

    Six new acylated anthocyanins (1-6) were isolated along with the three known congeners (7-9) from the fresh roots of red radishes (Raphanus sativus L.) cultivated by our group. Their chemical structures were elucidated by spectroscopic properties. Among the six new anthocyanins, the five constituents (1, 2, 4-6) were shown to contain the malonyl function at 6-OH in the glucopyranosyl residue linked to C-5 in the pelargonidin nucleus.

  2. A new acylated flavonol glycoside from Derris triofoliata.

    PubMed

    Xu, Lu-Rong; Wu, Jun; Zhang, Si

    2006-01-01

    A new acylated flavonol glycoside, kaempferol 3-O-[(6''''-feruloyl)-beta-D-glucopyranosyl-(1 --> 3)]-[alpha-L-rhamnopyranosyl-(1 --> 6)]-beta-D-glucopyranoside and two known cyclolignan glycosides, (+)-lyoniresinol-3alpha-O-beta-D-glucopyranoside and ( - )-lyoniresinol-3alpha-O-beta-D-glucopyranoside were isolated from n-BuOH extracts of the aerial parts of Derris triofoliata, their structures were determined from spectroscopic and chemical evidences.

  3. Hybrid Potential Simulation of the Acylation of Enterococcus faecium l,d-Transpeptidase by Carbapenems.

    PubMed

    Bhattacharjee, Nicholus; Field, Martin J; Simorre, Jean-Pierre; Arthur, Michel; Bougault, Catherine M

    2016-06-01

    The l,d-transpeptidases, Ldts, catalyze peptidoglycan cross-linking in β-lactam-resistant mutant strains of several bacteria, including Enterococcus faecium and Mycobacterium tuberculosis. Although unrelated to the essential d,d-transpeptidases, which are inactivated by the β-lactam antibiotics, they are nevertheless inhibited by the carbapenem antibiotics, making them potentially useful targets in the treatment of some important diseases. In this work, we have investigated the acylation mechanism of the Ldt from E. faecium by the carbapenem, ertapenem, using computational techniques. We have employed molecular dynamics simulations in conjunction with QC/MM hybrid potential calculations to map out possible reaction paths. We have focused on determining the following: (i) the protonation state of the nucleophilic cysteine of the enzyme when it attacks; (ii) whether nucleophilic attack and β-lactam ring-opening are concerted or stepwise, the latter occurring via an oxyanion intermediate; and (iii) the identities of the proton acceptors at the beginning and end of the reaction. Overall, we note that there is considerable plasticity in the mechanisms, owing to the significant flexibility of the enzyme, but find that the preferred pathways are ones in which nucleophilic attack of cysteine thiolate is concerted with β-lactam ring-opening. PMID:27196382

  4. Effects of short-chain acyl-CoA dehydrogenase on cardiomyocyte apoptosis.

    PubMed

    Zeng, Zhenhua; Huang, Qiuju; Shu, Zhaohui; Liu, Peiqing; Chen, Shaorui; Pan, Xuediao; Zang, Linquan; Zhou, Sigui

    2016-07-01

    Short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme of fatty acid β-oxidation, plays an important role in cardiac hypertrophy. However, its effect on the cardiomyocyte apoptosis remains unknown. We aimed to determine the role of SCAD in tert-butyl hydroperoxide (tBHP)-induced cardiomyocyte apoptosis. The mRNA and protein expression of SCAD were significantly down-regulated in the cardiomyocyte apoptosis model. Inhibition of SCAD with siRNA-1186 significantly decreased SCAD expression, enzyme activity and ATP content, but obviously increased the content of free fatty acids. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as cardiomyocytes treated with tBHP, such as the increase in cell apoptotic rate, the activation of caspase3 and the decrease in the Bcl-2/Bax ratio, which showed that SCAD may play an important role in primary cardiomyocyte apoptosis. The changes of phosphonate AMP-activated protein kinase α (p-AMPKα) and Peroxisome proliferator-activated receptor α (PPARα) in cardiomyocyte apoptosis were consistent with that of SCAD. Furthermore, PPARα activator fenofibrate and AMPKα activator AICAR treatment significantly increased the expression of SCAD and inhibited cardiomyocyte apoptosis. In conclusion, for the first time our findings directly demonstrated that SCAD may be as a new target to prevent cardiomyocyte apoptosis through the AMPK/PPARα/SCAD signal pathways. PMID:26989860

  5. Hybrid Potential Simulation of the Acylation of Enterococcus faecium l,d-Transpeptidase by Carbapenems.

    PubMed

    Bhattacharjee, Nicholus; Field, Martin J; Simorre, Jean-Pierre; Arthur, Michel; Bougault, Catherine M

    2016-06-01

    The l,d-transpeptidases, Ldts, catalyze peptidoglycan cross-linking in β-lactam-resistant mutant strains of several bacteria, including Enterococcus faecium and Mycobacterium tuberculosis. Although unrelated to the essential d,d-transpeptidases, which are inactivated by the β-lactam antibiotics, they are nevertheless inhibited by the carbapenem antibiotics, making them potentially useful targets in the treatment of some important diseases. In this work, we have investigated the acylation mechanism of the Ldt from E. faecium by the carbapenem, ertapenem, using computational techniques. We have employed molecular dynamics simulations in conjunction with QC/MM hybrid potential calculations to map out possible reaction paths. We have focused on determining the following: (i) the protonation state of the nucleophilic cysteine of the enzyme when it attacks; (ii) whether nucleophilic attack and β-lactam ring-opening are concerted or stepwise, the latter occurring via an oxyanion intermediate; and (iii) the identities of the proton acceptors at the beginning and end of the reaction. Overall, we note that there is considerable plasticity in the mechanisms, owing to the significant flexibility of the enzyme, but find that the preferred pathways are ones in which nucleophilic attack of cysteine thiolate is concerted with β-lactam ring-opening.

  6. Gastrointestinal uptake of nasunin, acylated anthocyanin in eggplant.

    PubMed

    Ichiyanagi, Takashi; Terahara, Norihiko; Rahman, M Mamunur; Konishi, Tetsuya

    2006-07-26

    We previously showed that nasunin, acylated anthocyanins in eggplant peel, comprises two isomers, cis-nasunin and trans-nasunin. In this study, gastrointestinal absorption of cis- and trans-nasunins was studied in rats. Orally administered nasunins were quickly absorbed in their original acylated forms and maximally appeared in blood plasma after 15 min. When the maximum plasma concentration and area under the plasma concentration curve were normalized by orally administered dose (micromoles per kilogram), there was no significant difference in the uptake efficiency between two isomers and both exhibited a plasma level almost identical to that of delphinidin 3-O-beta-D-glucopyranoside. However, metabolites such as 4'-O-methyl analogues and extended glucuronides which were observed for delphinidin 3-O-beta-D-glucopyranoside and cyanidin 3-O-beta-D-glucopyranoside metabolisms were not detected in urine or blood plasma. Moreover, deacylated and glycolytic products of nasunins such as delphinidin 3-O-beta-D-glucopyranoside or delphinidin (aglycone) were also not detected in blood plasma even after oral administration for 8 h. These results indicated that nasunins were absorbed in their original acylated forms and exhibit a bioavailability almost identical to that of nonacylated anthocyanins. PMID:16848510

  7. Naphthalene Derivatives Induce Acyl Chain Interdigitation in Dipalmitoylphosphatidylcholine Bilayers.

    PubMed

    Kamal, Md Arif; Raghunathan, V A

    2016-01-14

    The interdigitated phase of the lipid bilayer results when acyl chains from opposing monolayers fully interpenetrate such that the terminal methyl groups of the respective lipid chains are located at the interfacial region on the opposite sides of the bilayer. Usually, chain interdigitation is not encountered in a symmetric chain phosphatidylcholine (PC) membrane but can be induced under certain special conditions. In this article, we elucidate the contribution of small amphiphatic molecules in altering the physical properties of a symmetric chain PC bilayer membrane, which results in acyl chain interdigitation. Using small-angle X-ray scattering (SAXS), we have carried out a systematic investigation of the physical interactions of three naphthalene derivatives containing hydroxyl groups: β-naphthol, 2,3-dihydroxynaphthalene, and 2,7-dihydroxynaphthalene, with dipalmitoylphosphatidylcholine (DPPC) bilayers. On the basis of the diffraction patterns, we have determined the temperature-composition phase diagrams of these binary mixtures. The present study not only enables us to gain insight into the role played by small molecules in altering the packing arrangement of the acyl chains of the constituting PC lipids of the bilayer but also brings to light some important features that have not yet been reported hitherto. One such feature is the stabilization of the enigmatic asymmetric ripple phase over a wide temperature and concentration range. The results presented here strongly point toward a clear correlation between chain interdigitation and the stability of the ripple phase.

  8. Fatty acid acylation of salivary mucin in rat submandibular glands

    SciTech Connect

    Slomiany, B.L.; Murty, V.L.; Takagi, A.; Tsukada, H.; Kosmala, M.; Slomiany, A.

    1985-11-01

    The acylation of salivary mucin with fatty acids and its biosynthesis was investigated by incubating rat submandibular salivary gland cells with (/sup 3/H)palmitic acid and (/sup 3/H)proline. The elaborated extracellular and intracellular mucus glycoproteins following delipidation, Bio-Gel P-100 chromatography, and CsCl equilibrium density gradient centrifugation were analyzed for the distribution of the labeled tracers. The incorporation of both markers into mucus glycoprotein increased steadily with time up to 4 h, at which time about 65% of (/sup 3/H)palmitate and (/sup 3/H)proline were found in the extracellular glycoprotein and 35% in the intracellular glycoprotein. The incorporation ratio of proline/palmitate, while showing an increase with incubation time in the extracellular glycoprotein, remained essentially unchanged with time in the intracellular glycoprotein and at 4 h reached respective values of 0.14 and 1.12. The fact that the proline/palmitate incorporation ratio in the intracellular glycoprotein at 1 h of incubation was 22 times higher than in the extracellular and 8 times higher after 4 h suggests that acylation occurs intracellularly and that fatty acids are added after apomucin polypeptide synthesis. As the incorporation of palmitate within the intracellular mucin was greater in the mucus glycoprotein subunit, it would appear that fatty acid acylation of mucin subunits preceeds their assembly into the mucus glycoprotein polymer.

  9. Intestinal adaptation after massive intestinal resection

    PubMed Central

    Weale, A; Edwards, A; Bailey, M; Lear, P

    2005-01-01

    Patients with short bowel syndrome require long term parenteral nutrition support. However, after massive intestinal resection the intestine undergoes adaptation and nutritional autonomy may be obtained. Given that the complications of parenteral nutrition may be life threatening or result in treatment failure and the need for intestinal transplantation, a more attractive option is to wean patients off nutrition support by optimising the adaptive process. The article examines the evidence that after extensive small bowel resection adaptation occurs in humans and focuses on the factors that influence adaptation and the strategies that have been used to optimise this process. The review is based on an English language Medline search with secondary references obtained from key articles. There is evidence that adaptation occurs in humans. Adaptation is a complex process that results in response to nutrient and non-nutrient stimuli. Successful and reproducible strategies to improve adaptation remain elusive despite an abundance of experimental data. Nevertheless given the low patient survival and quality of life associated with other treatments for irreversible intestinal failure it is imperative that clinical research continues into the optimisation of the adaptation. PMID:15749794

  10. Intestinal colonization resistance

    PubMed Central

    Lawley, Trevor D; Walker, Alan W

    2013-01-01

    Dense, complex microbial communities, collectively termed the microbiota, occupy a diverse array of niches along the length of the mammalian intestinal tract. During health and in the absence of antibiotic exposure the microbiota can effectively inhibit colonization and overgrowth by invading microbes such as pathogens. This phenomenon is called ‘colonization resistance’ and is associated with a stable and diverse microbiota in tandem with a controlled lack of inflammation, and involves specific interactions between the mucosal immune system and the microbiota. Here we overview the microbial ecology of the healthy mammalian intestinal tract and highlight the microbe–microbe and microbe–host interactions that promote colonization resistance. Emerging themes highlight immunological (T helper type 17/regulatory T-cell balance), microbiota (diverse and abundant) and metabolic (short-chain fatty acid) signatures of intestinal health and colonization resistance. Intestinal pathogens use specific virulence factors or exploit antibiotic use to subvert colonization resistance for their own benefit by triggering inflammation to disrupt the harmony of the intestinal ecosystem. A holistic view that incorporates immunological and microbiological facets of the intestinal ecosystem should facilitate the development of immunomodulatory and microbe-modulatory therapies that promote intestinal homeostasis and colonization resistance. PMID:23240815

  11. Characterization of oryza sativa acyl activating enzyme3 (OsAAE3)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oxalate, the smallest of the dicarboxylic acids, is produced in many plants. This acid has been shown to play an important role in both plant physiology and defense, specifically in regards to metal detoxification, calcium regulation, sucking and chewing insect deterrence, and the production of calc...

  12. Intestinal disaccharidase activity in patients with autism: effect of age, gender, and intestinal inflammation.

    PubMed

    Kushak, Rafail I; Lauwers, Gregory Y; Winter, Harland S; Buie, Timothy M

    2011-05-01

    Intestinal disaccharidase activities were measured in 199 individuals with autism to determine the frequency of enzyme deficiency. All patients had duodenal biopsies that were evaluated morphologically and assayed for lactase, sucrase, and maltase activity. Frequency of lactase deficiency was 58% in autistic children ≤ 5 years old and 65% in older patients. As would be expected, patients with autism at age 5 > years demonstrated significant decline in lactase activity (24%, p = .02) in comparison with ≤ 5 years old autistic patients. Boys ≤ 5 years old with autism had 1.7 fold lower lactase activity than girls with autism (p = .02). Only 6% of autistic patients had intestinal inflammation. Lactase deficiency not associated with intestinal inflammation or injury is common in autistic children and may contribute to abdominal discomfort, pain and observed aberrant behavior. Most autistic children with lactose intolerance are not identified by clinical history. PMID:21415091

  13. Pediatric intestinal motility disorders

    PubMed Central

    Gfroerer, Stefan; Rolle, Udo

    2015-01-01

    Pediatric intestinal motility disorders affect many children and thus not only impose a significant impact on pediatric health care in general but also on the quality of life of the affected patient. Furthermore, some of these conditions might also have implications for adulthood. Pediatric intestinal motility disorders frequently present as chronic constipation in toddler age children. Most of these conditions are functional, meaning that constipation does not have an organic etiology, but in 5% of the cases, an underlying, clearly organic disorder can be identified. Patients with organic causes for intestinal motility disorders usually present in early infancy or even right after birth. The most striking clinical feature of children with severe intestinal motility disorders is the delayed passage of meconium in the newborn period. This sign is highly indicative of the presence of Hirschsprung disease (HD), which is the most frequent congenital disorder of intestinal motility. HD is a rare but important congenital disease and the most significant entity of pediatric intestinal motility disorders. The etiology and pathogenesis of HD have been extensively studied over the last several decades. A defect in neural crest derived cell migration has been proven as an underlying cause of HD, leading to an aganglionic distal end of the gut. Numerous basic science and clinical research related studies have been conducted to better diagnose and treat HD. Resection of the aganglionic bowel remains the gold standard for treatment of HD. Most recent studies show, at least experimentally, the possibility of a stem cell based therapy for HD. This editorial also includes rare causes of pediatric intestinal motility disorders such as hypoganglionosis, dysganglionosis, chronic intestinal pseudo-obstruction and ganglioneuromatosis in multiple endocrine metaplasia. Underlying organic pathologies are rare in pediatric intestinal motility disorders but must be recognized as early as

  14. Characterization of new glycolipid biosurfactants, tri-acylated mannosylerythritol lipids, produced by Pseudozyma yeasts.

    PubMed

    Fukuoka, Tokuma; Morita, Tomotake; Konishi, Masaaki; Imura, Tomohiro; Kitamoto, Dai

    2007-07-01

    Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by Pseudozyma yeasts. They show not only the excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from soybean oil by P. antarctica and P. rugulosa, some new extracellular glycolipids (more hydrophobic than the previously reported di-acylated MELs) were found in the culture medium. The most hydrophobic one was identified as 1-O-alka(e)noyl-4-O-[(4',6'-di-O-acetyl-2',3'-di-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol, namely tri-acylated MEL. Others were tri-acylated MELs bearing only one acetyl group. The tri-acylated MEL could be prepared by the lipase-catalyzed esterification of a di-acylated MEL with oleic acid implying that the new glycolipids are synthesized from di-acylated MELs in the culture medium containing the residual fatty acids. PMID:17417694

  15. Age-related increases in F344 rat intestine microsomal quercetin glucuronidation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to establish the extent age modifies intestinal quercetin glucuronidation capacity. Pooled microsomal fractions of three equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats (n=8/group) were employed to model the enzyme kinetics of UDP-gl...

  16. Effects of dietary plant meal and soya-saponin supplementation on intestinal and hepatic lipid droplet accumulation and lipoprotein and sterol metabolism in Atlantic salmon (Salmo salar L.).

    PubMed

    Gu, Min; Kortner, Trond M; Penn, Michael; Hansen, Anne Kristine; Krogdahl, Åshild

    2014-02-01

    Altered lipid metabolism has been shown in fish fed plant protein sources. The present study aimed to gain further insights into how intestinal and hepatic lipid absorption and metabolism are modulated by plant meal (PM) and soya-saponin (SA) inclusion in salmon feed. Post-smolt Atlantic salmon were fed for 10 weeks one of four diets based on fishmeal or PM, with or without 10 g/kg SA. PM inclusion resulted in decreased growth performance, excessive lipid droplet accumulation in the pyloric caeca and liver, and reduced plasma cholesterol levels. Intestinal and hepatic gene expression profiling revealed an up-regulation of the expression of genes involved in lipid absorption and lipoprotein (LP) synthesis (apo, fatty acid transporters, microsomal TAG transfer protein, acyl-CoA cholesterol acyltransferase, choline kinase and choline-phosphate cytidylyltransferase A), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase) and associated transcription factors (sterol regulatory element-binding protein 2 and PPARγ). SA inclusion resulted in reduced body pools of cholesterol and bile salts. The hepatic gene expression of the rate-limiting enzyme in bile acid biosynthesis (cytochrome P450 7A1 (cyp7a1)) as well as the transcription factor liver X receptor and the bile acid transporter abcb11 (ATP-binding cassette B11) was down-regulated by SA inclusion. A significant interaction was observed between PM inclusion and SA inclusion for plasma cholesterol levels. In conclusion, gene expression profiling suggested that the capacity for LP assembly and cholesterol synthesis was up-regulated by PM exposure, probably as a compensatory mechanism for excessive lipid droplet accumulation and reduced plasma cholesterol levels. SA inclusion had hypocholesterolaemic effects on Atlantic salmon, accompanied by decreased bile salt metabolism.

  17. The in vitro hydrolysis of phytosterol conjugates in food matrices by mammalian digestive enzymes.

    PubMed

    Moreau, Robert A; Hicks, Kevin B

    2004-08-01

    All fruits, vegetables, and grains contain phytosterols. Numerous clinical studies have documented that phytosterols lower LDL-cholesterol levels and thereby reduce the risk of cardiovascular disease. Most experts believe that the cholesterol-lowering mechanism of phytosterols requires that they be in their "free" form. In addition to their occurrence in the free form, phytosterols also occur as four common phytosterol conjugates: (i) fatty acyl esters, (ii) hydroxycinnamate esters, (iii) steryl glycosides, and (iv) fatty acylated steryl glycosides. This study was undertaken to investigate the extent of hydrolysis of four common phytosterol conjugates by mammalian digestive enzymes (cholesterol esterase and pancreatin, a mixture of pancreatic enzymes) and for comparison purposes, by KOH. Two types of purified hydroxycinnamate esters (sitostanyl ferulate and oryzanol, a mixture of hydroxycinnamate esters purified from rice bran oil) were hydrolyzed by cholesterol esterase and by pancreatin. Both cholesterol esterase and pancreatin hydrolyzed the phytosteryl esters in two functional food matrices, and they hydrolyzed the hydroxycinnamate esters in corn fiber oil. This is the first report to demonstrate that phytostanyl ferulate esters (which are present at levels of 3-6% in corn fiber oil) are hydrolyzed by pancreatic cholesterol esterase. It is also the first report that pancreatin contains enzymes that hydrolyze the fatty acyl moiety of fatty acylated steryl glycoside, converting it to steryl glycoside. Pancreatin had no effect on steryl glycosides. The ability of pancreatin to hydrolyze three other types of lipid conjugates was also evaluated. Phospholipids were completely hydrolyzed. About half of the galactolipids were hydrolyzed, and less than 10% of the polyamine conjugates were hydrolyzed. The extents of hydrolysis of phytosteryl esters by base (saponification) were also studied, and conditions commonly used for the saponification of acyl lipids (1.5 N

  18. A new role for penicillin acylases: degradation of acyl homoserine lactone quorum sensing signals by Kluyvera citrophila penicillin G acylase.

    PubMed

    Mukherji, Ruchira; Varshney, Nishant Kumar; Panigrahi, Priyabrata; Suresh, C G; Prabhune, Asmita

    2014-03-01

    Use of penicillin acylases for the production of semi-synthetic penicillins is well-known. Escherichia coli penicillin G acylase (EcPGA) has been extensively used for this purpose; however, Kluyvera citrophila penicillin G acylase (KcPGA) is assumed to be a better substitute, owing to its increased resilience to extreme pH conditions and ease of immobilization. In the present article we report a new dimension for the amidase activity of KcPGA by demonstrating its ability to cleave bacterial quorum sensing signal molecules, acyl homoserine lactones (AHL) with acyl chain length of 6-8 with or without oxo-substitution at third carbon position. Initial evidence of AHL degrading capability of KcPGA was obtained using CV026 based bioassay method. Kinetic studies performed at pH 8.0 and 50 °C revealed 3-oxo-C6 HSL to be the best substrate for the enzyme with V(max) and K(m) values of 21.37+0.85 mM/h/mg of protein and 0.1+0.01 mM, respectively. C6 HSL was found to be the second best substrate with V(max) and K(m) value of 10.06+0.27 mM/h/mg of protein and 0.28+0.02 mM, respectively. Molecular modeling and docking studies performed on the active site of the enzyme support these findings by showing the fitting of AHLs perfectly within the hydrophobic pocket of the enzyme active site.

  19. Intestinal pseudo-obstruction

    MedlinePlus

    ... Taking drugs that slow intestinal movements. These include narcotic (pain) medicines and drugs used when you are ... that may have caused the problem (such as narcotic drugs) may help. In severe cases, surgery may ...

  20. Small intestine (image)

    MedlinePlus

    The small intestine is the portion of the digestive system most responsible for absorption of nutrients from food into the bloodstream. The pyloric sphincter governs the passage of partly digested food ...

  1. Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.

    PubMed

    Grant, Christa N; Mojica, Salvador Garcia; Sala, Frederic G; Hill, J Ryan; Levin, Daniel E; Speer, Allison L; Barthel, Erik R; Shimada, Hiroyuki; Zachos, Nicholas C; Grikscheit, Tracy C

    2015-04-15

    Short bowel syndrome (SBS) is a devastating condition in which insufficient small intestinal surface area results in malnutrition and dependence on intravenous parenteral nutrition. There is an increasing incidence of SBS, particularly in premature babies and newborns with congenital intestinal anomalies. Tissue-engineered small intestine (TESI) offers a therapeutic alternative to the current standard treatment, intestinal transplantation, and has the potential to solve its biggest challenges, namely donor shortage and life-long immunosuppression. We have previously demonstrated that TESI can be generated from mouse and human small intestine and histologically replicates key components of native intestine. We hypothesized that TESI also recapitulates native small intestine function. Organoid units were generated from mouse or human donor intestine and implanted into genetically identical or immunodeficient host mice. After 4 wk, TESI was harvested and either fixed and paraffin embedded or immediately subjected to assays to illustrate function. We demonstrated that both mouse and human tissue-engineered small intestine grew into an appropriately polarized sphere of intact epithelium facing a lumen, contiguous with supporting mesenchyme, muscle, and stem/progenitor cells. The epithelium demonstrated major ultrastructural components, including tight junctions and microvilli, transporters, and functional brush-border and digestive enzymes. This study demonstrates that tissue-engineered small intestine possesses a well-differentiated epithelium with intact ion transporters/channels, functional brush-border enzymes, and similar ultrastructural components to native tissue, including progenitor cells, whether derived from mouse or human cells. PMID:25573173

  2. Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.

    PubMed

    Grant, Christa N; Mojica, Salvador Garcia; Sala, Frederic G; Hill, J Ryan; Levin, Daniel E; Speer, Allison L; Barthel, Erik R; Shimada, Hiroyuki; Zachos, Nicholas C; Grikscheit, Tracy C

    2015-04-15

    Short bowel syndrome (SBS) is a devastating condition in which insufficient small intestinal surface area results in malnutrition and dependence on intravenous parenteral nutrition. There is an increasing incidence of SBS, particularly in premature babies and newborns with congenital intestinal anomalies. Tissue-engineered small intestine (TESI) offers a therapeutic alternative to the current standard treatment, intestinal transplantation, and has the potential to solve its biggest challenges, namely donor shortage and life-long immunosuppression. We have previously demonstrated that TESI can be generated from mouse and human small intestine and histologically replicates key components of native intestine. We hypothesized that TESI also recapitulates native small intestine function. Organoid units were generated from mouse or human donor intestine and implanted into genetically identical or immunodeficient host mice. After 4 wk, TESI was harvested and either fixed and paraffin embedded or immediately subjected to assays to illustrate function. We demonstrated that both mouse and human tissue-engineered small intestine grew into an appropriately polarized sphere of intact epithelium facing a lumen, contiguous with supporting mesenchyme, muscle, and stem/progenitor cells. The epithelium demonstrated major ultrastructural components, including tight junctions and microvilli, transporters, and functional brush-border and digestive enzymes. This study demonstrates that tissue-engineered small intestine possesses a well-differentiated epithelium with intact ion transporters/channels, functional brush-border enzymes, and similar ultrastructural components to native tissue, including progenitor cells, whether derived from mouse or human cells.

  3. ER stress transcription factor Xbp1 suppresses intestinal tumorigenesis and directs intestinal stem cells

    PubMed Central

    Niederreiter, Lukas; Fritz, Teresa M.J.; Adolph, Timon E.; Krismer, Anna-Maria; Offner, Felix A.; Tschurtschenthaler, Markus; Flak, Magdalena B.; Hosomi, Shuhei; Tomczak, Michal F.; Kaneider, Nicole C.; Sarcevic, Edina; Kempster, Sarah L.; Raine, Tim; Esser, Daniela; Rosenstiel, Philip; Kohno, Kenji; Iwawaki, Takao; Tilg, Herbert

    2013-01-01

    Unresolved endoplasmic reticulum (ER) stress in the epithelium can provoke intestinal inflammation. Hypomorphic variants of ER stress response mediators, such as X-box–binding protein 1 (XBP1), confer genetic risk for inflammatory bowel disease. We report here that hypomorphic Xbp1 function instructs a multilayered regenerative response in the intestinal epithelium. This is characterized by intestinal stem cell (ISC) expansion as shown by an inositol-requiring enzyme 1α (Ire1α)–mediated increase in Lgr5+ and Olfm4+ ISCs and a Stat3-dependent increase in the proliferative output of transit-amplifying cells. These consequences of hypomorphic Xbp1 function are associated with an increased propensity to develop colitis-associated and spontaneous adenomatous polyposis coli (APC)–related tumors of the intestinal epithelium, which in the latter case is shown to be dependent on Ire1α. This study reveals an unexpected role for Xbp1 in suppressing tumor formation through restraint of a pathway that involves an Ire1α- and Stat3-mediated regenerative response of the epithelium as a consequence of ER stress. As such, Xbp1 in the intestinal epithelium not only regulates local inflammation but at the same time also determines the propensity of the epithelium to develop tumors. PMID:24043762

  4. Regulation of gene expression through a transcriptional repressor that senses acyl-chain length in membrane phospholipids.

    PubMed

    Hofbauer, Harald F; Schopf, Florian H; Schleifer, Hannes; Knittelfelder, Oskar L; Pieber, Bartholomäus; Rechberger, Gerald N; Wolinski, Heimo; Gaspar, Maria L; Kappe, C Oliver; Stadlmann, Johannes; Mechtler, Karl; Zenz, Alexandra; Lohner, Karl; Tehlivets, Oksana; Henry, Susan A; Kohlwein, Sepp D

    2014-06-23

    Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription.

  5. Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

    SciTech Connect

    Ghadbane, Hemza; Brown, Alistair K.; Kremer, Laurent; Besra, Gurdyal S. Fütterer, Klaus

    2007-10-01

    Binding of Ni{sup 2+} ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution. Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni{sup 2+} ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

  6. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  7. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  8. Molecular dynamics simulations of class C beta-lactamase from Citrobacter freundii: insights into the base catalyst for acylation.

    PubMed

    Díaz, Natalia; Suárez, Dimas; Sordo, Tomás L

    2006-01-17

    Herein, we present results from molecular dynamics (MD) simulations of the class C beta-lactamase from Citrobacter freundii and its Michaelis complex with aztreonam. Four different configurations of the active site were modeled in aqueous solution, and their relative stability was estimated by means of quantum mechanical energy calculations. For the free enzyme, the energetically most stable configurations present a neutral Lys67 residue or an anionic Tyr150 side chain. Our calculations predict that these two configurations are quite close in terms of free energy, the anionic Tyr150 state being favored by approximately 1 kcal/mol. In contrast, for the noncovalent complex formed between the C. freundii enzyme and aztreonam, the energetic analyses predict that the configuration with the neutral Lys67 residue is much more stable than the anionic Tyr150 one (approximately 20 kcal/mol). Moreover, the MD simulations reveal that the neutral Lys67 state results in a proper enzyme-aztreonam orientation for nucleophilic attack and in a very stable contact between the nucleophilic hydroxyl group of Ser64 and the neutral amino side chain of Lys67. Thus, both the computed free energies and the structural analyses support the assignation of Lys67 as the base catalyst for the acylation step in the native form of the C. freundii enzyme.

  9. Acyl-Protein Thioesterase 2 Catalizes the Deacylation of Peripheral Membrane-Associated GAP-43

    PubMed Central

    Tomatis, Vanesa M.; Trenchi, Alejandra; Gomez, Guillermo A.; Daniotti, Jose L.

    2010-01-01

    An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution. PMID:21152083

  10. Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase.

    PubMed

    Chung, Jiwoung; Goo, Eunhye; Yu, Sangheon; Choi, Okhee; Lee, Jeehyun; Kim, Jinwoo; Kim, Hongsup; Igarashi, Jun; Suga, Hiroaki; Moon, Jae Sun; Hwang, Ingyu; Rhee, Sangkee

    2011-07-19

    Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5'-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents.

  11. The intestine is a blender

    NASA Astrophysics Data System (ADS)

    Yang, Patricia; Lamarca, Morgan; Kravets, Victoria; Hu, David

    According to the U.S. Department of Health and Human Services, digestive disease affects 60 to 70 million people and costs over 140 billion annually. Despite the significance of the gastrointestinal tract to human health, the physics of digestion remains poorly understood. In this study, we ask a simple question: what sets the frequency of intestinal contractions? We measure the frequency of intestinal contractions in rats, as a function of distance down the intestine. We find that intestines Contract radially ten times faster than longitudinally. This motion promotes mixing and, in turn, absorption of food products by the intestinal wall. We calculate viscous dissipation in the intestinal fluid to rationalize the relationship between frequency of intestinal contraction and the viscosity of the intestinal contents. Our findings may help to understand the evolution of the intestine as an ideal mixer.

  12. The intestine is a blender

    NASA Astrophysics Data System (ADS)

    Yang, Patricia; Lamarca, Morgan; Hu, David

    2015-11-01

    According to the U.S. Department of Health and Human Services, digestive disease affects 60 to 70 million people and costs over 140 billion annually. Despite the significance of the gastrointestinal tract to human health, the physics of digestion remains poorly understood. In this study, we ask a simple question: what sets the frequency of intestinal contractions? We measure the frequency of intestinal contractions in rats, as a function of distance down the intestine. We find that intestines contract radially ten times faster than longitudinally. This motion promotes mixing and, in turn, absorption of food products by the intestinal wall. We calculate viscous dissipation in the intestinal fluid to rationalize the relationship between frequency of intestinal contraction and the viscosity of the intestinal contents. Our findings may help to understand the evolution of the intestine as an ideal mixer.

  13. Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed.

    PubMed

    Metz, J G; Pollard, M R; Anderson, L; Hayes, T R; Lassner, M W

    2000-03-01

    The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes.

  14. Purification of a Jojoba Embryo Fatty Acyl-Coenzyme A Reductase and Expression of Its cDNA in High Erucic Acid Rapeseed

    PubMed Central

    Metz, James G.; Pollard, Michael R.; Anderson, Lana; Hayes, Thomas R.; Lassner, Michael W.

    2000-01-01

    The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes. PMID:10712526

  15. An Ultrahigh Resolution Structure of TEM-1 beta-Lactamase Suggests a Role for Glu166 as the General Base in Acylation

    SciTech Connect

    Minasov, George; Wang, Xiaojun; Shoichet, Brian K.

    2010-03-08

    Although TEM-1 {beta}-lactamase is among the best studied enzymes, its acylation mechanism remains controversial. To investigate this problem, the structure of TEM-1 in complex with an acylation transition-state analogue was determined at ultrahigh resolution (0.85 {angstrom}) by X-ray crystallography. The quality of the data was such as to allow for refinement to an R-factor of 9.1% and an R{sub free} of 11.2%. In the resulting structure, the electron density features were clear enough to differentiate between single and double bonds in carboxylate groups, to identify multiple conformations that are occupied by residues and loops, and to assign 70% of the protons in the protein. Unexpectedly, even at pH 8.0 where the protein was crystallized, the active site residue Glu166 is clearly protonated. This supports the hypothesis that Glu166 is the general base in the acylation half of the reaction cycle. This structure suggests that Glu166 acts through the catalytic water to activate Ser70 for nucleophilic attack on the {beta}-lactam ring of the substrate. The hydrolytic mechanism of class A {beta}-lactamases, such as TEM-1, appears to be symmetrical, as are the serine proteases. Apart from its mechanistic implications, this atomic resolution structure affords an unusually detailed view of the structure, dynamics, and hydrogen-bonding networks of TEM-1, which may be useful for the design of inhibitors against this key antibiotic resistance target.

  16. Fatty acid biosynthesis in Pseudomonas aeruginosa is initiated by the FabY class of β-ketoacyl acyl carrier protein synthases.

    PubMed

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A; Meredith, Timothy C

    2012-10-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes.

  17. 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.

  18. Intestinal Diffusion Barrier: Unstirred Water Layer or Membrane Surface Mucous Coat?

    NASA Astrophysics Data System (ADS)

    Smithson, Kenneth W.; Millar, David B.; Jacobs, Lucien R.; Gray, Gary M.

    1981-12-01

    The dimensions of the small intestinal diffusion barrier interposed between luminal nutrients and their membrane receptors were determined from kinetic analysis of substrate hydrolysis by integral surface membrane enzymes. The calculated equivalent thickness of the unstirred water layer was too large to be compatible with the known dimensions of rat intestine. The discrepancy could be reconciled by consideration of the mucous coat overlying the intestinal surface membrane. Integral surface membrane proteins could not be labeled by an iodine-125 probe unless the surface coat was first removed. The mucoprotein surface coat appears to constitute an important diffusion barrier for nutrients seeking their digestive and transport sites on the outer intestinal membrane.

  19. Acylation of Antioxidant of Bamboo Leaves with Fatty Acids by Lipase and the Acylated Derivatives' Efficiency in the Inhibition of Acrylamide Formation in Fried Potato Crisps.

    PubMed

    Ma, Xiang; Wang, Erpei; Lu, Yuyun; Wang, Yong; Ou, Shiyi; Yan, Rian

    2015-01-01

    This study selectively acylated the primary hydroxyl groups on flavonoids in antioxidant of bamboo leaves (AOB) using lauric acid with Candida antarctica lipase B in tert-amyl-alcohol. The separation and isolation of acylated derivatives were performed using silica gel column chromatography with a mixture of dichloromethane/diethyl ether/methanol as eluents. Both thin layer chromatography and high-performance liquid chromatography analyses confirmed the high efficiency of the isolation process with the purified orientin-6″-laurate, isoorientin-6″-laurate, vitexin-6″-laurate, and isovitexin-6″-laurate that were obtained. The addition of AOB and acylated AOB reduced acrylamide formation in fried potato crisps. Results showed that 0.05% AOB and 0.05% and 0.1% acylated AOB groups significantly (p < 0.05) reduced the content of acrylamide in potato crisps by 30.7%, 44.5%, and 46.9%, respectively. PMID:26098744

  20. Acylation of Antioxidant of Bamboo Leaves with Fatty Acids by Lipase and the Acylated Derivatives’ Efficiency in the Inhibition of Acrylamide Formation in Fried Potato Crisps

    PubMed Central

    Ma, Xiang; Wang, Erpei; Lu, Yuyun; Wang, Yong; Ou, Shiyi; Yan, Rian

    2015-01-01

    This study selectively acylated the primary hydroxyl groups on flavonoids in antioxidant of bamboo leaves (AOB) using lauric acid with Candida antarctica lipase B in tert-amyl-alcohol. The separation and isolation of acylated derivatives were performed using silica gel column chromatography with a mixture of dichloromethane/diethyl ether/methanol as eluents. Both thin layer chromatography and high-performance liquid chromatography analyses confirmed the high efficiency of the isolation process with the purified orientin-6″-laurate, isoorientin-6″-laurate, vitexin-6″-laurate, and isovitexin-6″-laurate that were obtained. The addition of AOB and acylated AOB reduced acrylamide formation in fried potato crisps. Results showed that 0.05% AOB and 0.05% and 0.1% acylated AOB groups significantly (p < 0.05) reduced the content of acrylamide in potato crisps by 30.7%, 44.5%, and 46.9%, respectively. PMID:26098744

  1. Ginsenoside Rb1 protects the intestinal mucosal barrier following peritoneal air exposure

    PubMed Central

    Zhou, Feng; Zhang, Peichen; Chen, Xiaoxi; Yan, Jingyi; Yao, Jiangao; Yu, Zhen; Chen, Xiaolei

    2016-01-01

    Ginsenoside Rb1 (GRb1), which is one of the main ingredients derived from Panax ginseng, has been widely used to treat various gastrointestinal disorders. The present study aimed to determine whether GRb1 was able to prevent intestinal mucosal barrier damage in rats following peritoneal air exposure for 3 h. GRb1 (5, 10, and 20 mg/kg) was orally administrated via gavage four times prior to and following surgery. Blood and terminal ileum were sampled 24 h following surgery. Levels of serum D-lactate (D-LA) were detected using an enzyme-linked immunosorbent assay kit. Intestinal permeability was assessed by determining the intestinal clearance of fluorescein isothiocyanate-dextran (FD4). Activity of intestinal myeloperoxidase was measured to assess intestinal inflammation, and intestinal histopathology was assessed by light microscopy. The results showed that GRb1 reduced the level of serum D-LA, intestinal clearance of FD4, and the activity of intestinal myeloperoxidase. Intestinal edema and inflammation were also ameliorated by GRb1, and the Chiu's scores employed for assessing intestinal mucosal damage were also reduced in the GRb1-treated peritoneal air exposure group. In addition, GRb1 induced a significant difference at 10 and 20 mg/kg, indicating a dose-dependent effect. The results of the present study suggest that GRb1 may be able to protect the intestinal mucosal barrier against damage induced by peritoneal air exposure, which may be associated with its anti-inflammatory action. PMID:27703510

  2. Evaluation of Clopidogrel Conjugation Metabolism: PK Studies in Man and Mice of Clopidogrel Acyl Glucuronide.

    PubMed

    Savu, Simona Nicoleta; Silvestro, Luigi; Surmeian, Mariana; Remis, Lina; Rasit, Yuksel; Savu, Simona Rizea; Mircioiu, Constantin

    2016-09-01

    The existence of a glucuronide conjugate of the major circulating clopidogrel metabolites, called clopidogrel acyl glucuronide (CAG), is already known. However, information regarding its pharmacokinetics (PK), metabolism, and clearance are modest. We investigated in vivo the potential CAG trans-esterification to clopidogrel (reaction occurring in vitro in particular conditions) by administering the metabolite to mice. Experiments were then carried out on men, clopidogrel administered alone or followed by activated charcoal intake (intestinal reabsorption blockade). Study objectives included: PK comparison of CAG, clopidogrel carboxylic acid (CCA), and clopidogrel in plasma, determination of their elimination patterns in urine and feces, and tracking of charcoal-induced changes in PK and/or urinary excretion that would indicate relevant enterohepatic recycling of CAG. In mice, CAG was rapidly hydrolyzed to CCA after oral administration, whereas by intravenous route metabolic conversion to CCA was delayed. No levels of clopidogrel were detected in mice plasma, excluding any potential trans-esterification or other form of back-conversion in vivo. PK experiments in man showed that CAG is hydrolyzed in the gastrointestinal tract (very low concentrations in feces), but there is no evidence of enterohepatic recirculation. Quantitation of the three moieties in stool samples accounted for only 1.2% of an administered dose, suggesting that other yet unknown metabolites/degradation products formed through metabolic processes and/or the activity of local microflora are mainly excreted by this route. In man CAG was confirmed as one of the major terminal metabolites of clopidogrel, with a PK behavior similar to CCA. PMID:27402727

  3. Ortho C-H Acylation of Aryl Iodides by Palladium/Norbornene Catalysis.

    PubMed

    Dong, Zhe; Wang, Jianchun; Ren, Zhi; Dong, Guangbin

    2015-10-19

    Reported herein is a palladium/norbornene-catalyzed ortho-arene acylation of aryl iodides by a Catellani-type C-H functionalization. This transformation is enabled by isopropyl carbonate anhydrides, which serve as both an acyl cation equivalent and a hydride source.

  4. Structural properties of pepsin-solubilized collagen acylated by lauroyl chloride along with succinic anhydride.

    PubMed

    Li, Conghu; Tian, Zhenhua; Liu, Wentao; Li, Guoying

    2015-10-01

    The structural properties of pepsin-solubilized calf skin collagen acylated by lauroyl chloride along with succinic anhydride were investigated in this paper. Compared with native collagen, acylated collagen retained the unique triple helix conformation, as determined by amino acid analysis, circular dichroism and X-ray diffraction. Meanwhile, the thermostability of acylated collagen using thermogravimetric measurements was enhanced as the residual weight increased by 5%. With the temperature increased from 25 to 115 °C, the secondary structure of native and acylated collagens using Fourier transform infrared spectroscopy measurements was destroyed since the intensity of the major amide bands decreased and the positions of the major amide bands shifted to lower wavenumber, respectively. Meanwhile, two-dimensional correlation spectroscopy revealed that the most sensitive bands for acylated and native collagens were amide I and II bands, respectively. Additionally, the corresponding order of the groups between native and acylated collagens was different and the correlation degree for acylated collagen was weaker than that of native collagen, suggesting that temperature played a small influence on the conformation of acylated collagen, which might be concluded that the hydrophobic interaction improved the thermostability of collagen.

  5. Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Produces N-Acyl-Homoserine Lactone Autoinducers

    PubMed Central

    Bottomley, Peter J.

    2015-01-01

    Nitrobacter winogradskyi is a chemolithotrophic bacterium that plays a role in the nitrogen cycle by oxidizing nitrite to nitrate. Here, we demonstrate a functional N-acyl-homoserine lactone (acyl-HSL) synthase in this bacterium. The N. winogradskyi genome contains genes encoding a putative acyl-HSL autoinducer synthase (nwi0626, nwiI) and a putative acyl-HSL autoinducer receptor (nwi0627, nwiR) with amino acid sequences 38 to 78% identical to those in Rhodopseudomonas palustris and other Rhizobiales. Expression of nwiI and nwiR correlated with acyl-HSL production during culture. N. winogradskyi produces two distinct acyl-HSLs, N-decanoyl-l-homoserine lactone (C10-HSL) and a monounsaturated acyl-HSL (C10:1-HSL), in a cell-density- and growth phase-dependent manner, during batch and chemostat culture. The acyl-HSLs were detected by bioassay and identified by ultraperformance liquid chromatography with information-dependent acquisition mass spectrometry (UPLC-IDA-MS). The C=C bond in C10:1-HSL was confirmed by conversion into bromohydrin and detection by UPLC-IDA-MS. PMID:26092466

  6. Synthesis of photoactivatable azido-acyl caged oxazine fluorophores for live-cell imaging.

    PubMed

    Anzalone, Andrew V; Chen, Zhixing; Cornish, Virginia W

    2016-07-19

    We report the design and synthesis of a photoactivatable azido-acyl oxazine fluorophore. Photoactivation is achieved cleanly and rapidly with UV light, producing a single fluorescent oxazine photoproduct. We demonstrate the utility of azido-acyl caged oxazines for protein specific labeling in living mammalian cells using the TMP-tag technology. PMID:27377037

  7. Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Produces N-Acyl-Homoserine Lactone Autoinducers.

    PubMed

    Mellbye, Brett L; Bottomley, Peter J; Sayavedra-Soto, Luis A

    2015-09-01

    Nitrobacter winogradskyi is a chemolithotrophic bacterium that plays a role in the nitrogen cycle by oxidizing nitrite to nitrate. Here, we demonstrate a functional N-acyl-homoserine lactone (acyl-HSL) synthase in this bacterium. The N. winogradskyi genome contains genes encoding a putative acyl-HSL autoinducer synthase (nwi0626, nwiI) and a putative acyl-HSL autoinducer receptor (nwi0627, nwiR) with amino acid sequences 38 to 78% identical to those in Rhodopseudomonas palustris and other Rhizobiales. Expression of nwiI and nwiR correlated with acyl-HSL production during culture. N. winogradskyi produces two distinct acyl-HSLs, N-decanoyl-l-homoserine lactone (C10-HSL) and a monounsaturated acyl-HSL (C10:1-HSL), in a cell-density- and growth phase-dependent manner, during batch and chemostat culture. The acyl-HSLs were detected by bioassay and identified by ultraperformance liquid chromatography with information-dependent acquisition mass spectrometry (UPLC-IDA-MS). The C=C bond in C10:1-HSL was confirmed by conversion into bromohydrin and detection by UPLC-IDA-MS.

  8. Turnover of the 4'-phosphopantetheine prosthetic group of acyl carrier protein.

    PubMed

    Jackowski, S; Rock, C O

    1984-02-10

    Acyl carrier protein is an essential cofactor in fatty acid biosynthesis, and in contrast to the stability of the protein moiety during growth, its 4'-phosphopantetheine prosthetic group is metabolically active. The biosynthetic incorporation of deuterium into nonexchangeable positions of acyl carrier protein was found to enhance the sensitivity of the protein to pH-induced hydrodynamic expansion. This constitutional isotope effect was exploited to separate deuterated from normal acyl carrier protein by conformationally sensitive gel electrophoresis, thus providing the analytical framework for separating pre-existing (deuterated) from newly synthesized acyl carrier protein in pulse-chase experiments. The rate of acyl carrier protein prosthetic group turnover was found to depend on the intracellular concentration of coenzyme A. At low coenzyme A levels, prosthetic group turnover was four times faster than the rate of new acyl carrier protein biosynthesis but at the higher coenzyme A concentrations characteristic of logarithmic growth, turnover was an order of magnitude slower, amounting to approximately 25% of the acyl carrier protein pool per generation. These observations suggest that the acyl carrier protein prosthetic group turnover cycle may be related to coenzyme A metabolism rather than to lipid biosynthesis.

  9. Intestinal and multivisceral transplantation

    PubMed Central

    Meira, Sérgio Paiva; Guardia, Bianca Della; Evangelista, Andréia Silva; Matielo, Celso Eduardo Lourenço; Neves, Douglas Bastos; Pandullo, Fernando Luis; Felga, Guilherme Eduardo Gonçalves; Alves, Jefferson André da Silva; Curvelo, Lilian Amorim; Diaz, Luiz Gustavo Guedes; Rusi, Marcela Balbo; Viveiros, Marcelo de Melo; de Almeida, Marcio Dias; Epstein, Marina Gabrielle; Pedroso, Pamella Tung; Salvalaggio, Paolo; Meirelles, Roberto Ferreira; Rocco, Rodrigo Andrey; de Almeida, Samira Scalso; de Rezende, Marcelo Bruno

    2015-01-01

    Intestinal transplantation has shown exceptional growth over the past 10 years. At the end of the 1990’s, intestinal transplantation moved out of the experimental realm to become a routine practice in treating patients with severe complications related to total parenteral nutrition and intestinal failure. In the last years, several centers reported an increasing improvement in survival outcomes (about 80%), during the first 12 months after surgery, but long-term survival is still a challenge. Several advances led to clinical application of transplants. Immunosuppression involved in intestinal and multivisceral transplantation was the biggest gain for this procedure in the past decade due to tacrolimus, and new inducing drugs, mono- and polyclonal anti-lymphocyte antibodies. Despite the advancement of rigid immunosuppression protocols, rejection is still very frequent in the first 12 months, and can result in long-term graft loss. The future of intestinal transplantation and multivisceral transplantation appears promising. The major challenge is early recognition of acute rejection in order to prevent graft loss, opportunistic infections associated to complications, post-transplant lymphoproliferative disease and graft versus host disease; and consequently, improve results in the long run. PMID:25993080

  10. Genetics Home Reference: peroxisomal acyl-CoA oxidase deficiency

    MedlinePlus

    ... enzyme is found in sac-like cell structures (organelles) called peroxisomes, which contain a variety of enzymes ... 1 link) Health Topic: Leukodystrophies Genetic and Rare Diseases Information Center (1 link) Pseudoneonatal adrenoleukodystrophy Educational Resources ( ...

  11. Enzyme Kinetics

    PubMed Central

    Lam, C. F.; Priest, D. G.

    1972-01-01

    One of the most generally applicable algorithms for the derivation of steady-state rate equations for complex enzyme reaction mechanisms is that of King and Altman. Several modifications of this algorithm have been suggested; however, each requires the generation of numerous valid and invalid patterns and the subsequent elimination of those that are invalid. A method is presented, employing topological theory of linear graphs, for the systematic generation of only those patterns which are valid. This method is readily adaptable to use on a digital computer. An independent method for the calculation of the number of valid patterns is also presented. This calculation can be used to substantiate the accuracy of the patterns obtained. This calculation is also adaptable to computerization. Examples are included to demonstrate both the generation of patterns and the calculation of their number for specific enzyme mechanisms. PMID:5016111

  12. Alkylating enzymes.

    PubMed

    Wessjohann, Ludger A; Keim, Jeanette; Weigel, Benjamin; Dippe, Martin

    2013-04-01

    Chemospecific and regiospecific modifications of natural products by methyl, prenyl, or C-glycosyl moieties are a challenging and cumbersome task in organic synthesis. Because of the availability of an increasing number of stable and selective transferases and cofactor regeneration processes, enzyme-assisted strategies turn out to be promising alternatives to classical synthesis. Two categories of alkylating enzymes become increasingly relevant for applications: firstly prenyltransferases and terpene synthases (including terpene cyclases), which are used in the production of terpenoids such as artemisinin, or meroterpenoids like alkylated phenolics and indoles, and secondly methyltransferases, which modify flavonoids and alkaloids to yield products with a specific methylation pattern such as 7-O-methylaromadendrin and scopolamine.

  13. Role of adenosine 5'-monophosphate-activated protein kinase in α-linolenic acid-induced intestinal lipid metabolism.

    PubMed

    Zhou, Xihong; Chen, Jingqing; Wu, Weiche; Wang, Xinxia; Wang, Yizhen

    2015-09-28

    n-3 Long-chain PUFA up-regulate intestinal lipid metabolism. However, whether these metabolic effects of PUFA on intestine are mediated by AMP-activated protein kinase (AMPK) remains to be elucidated. To determine the effects of α-linolenic acid (ALA) on intestinal fatty acid (FA) metabolism and whether these effects were affected by AMPK deletion, mice deficient in the catalytic subunit of AMPKα1 or AMPKα2 and wild-type (WT) mice were fed either a high-fat diet (HF) or HF supplemented with ALA (HF-A). The results showed that ALA supplementation decreased serum TAG content in WT mice. ALA also increased mRNA expression of genes (carnitine palmitoyltransferase 1a, acyl-CoA oxidase 1, medium-chain acyl-CoA dehydrogenase, cytochrome P450 4A10 and pyruvate dehydrogenase kinase isoenzyme 4a) involved in intestinal lipid oxidation and mRNA expression of TAG synthesis-related genes (monoacylglycerol O-acyltransferase 2, diacylglycerol O-acyltransferases 1 and 2) in WT mice. Consistent with these, expression levels of phosphorylated AMPKα1 and AMPKα2 were also increased in WT mice after ALA addition. However, in the absence of either AMPKα1 or AMPKα2, ALA supplementation failed to increase intestinal lipid oxidation. In addition, no significant effects of either diet (HF and HF-A) or genotype (WT, AMPKα1(-/-) and AMPKα2(-/-)) on FA uptake in the intestine and faecal TAG output were observed. Our results suggest that AMPK is indispensable for the effects of ALA on intestinal lipid oxidation. PMID:26268732

  14. Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene.

    PubMed

    Knutzon, D S; Thompson, G A; Radke, S E; Johnson, W B; Knauf, V C; Kridl, J C

    1992-04-01

    Molecular gene transfer techniques have been used to engineer the fatty acid composition of Brassica rapa and Brassica napus (canola) oil. Stearoyl-acyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis, converting stearoyl-ACP to oleoyl-ACP. Seed-specific antisense gene constructs of B. rapa stearoyl-ACP desaturase were used to reduce the protein concentration and enzyme activity of stearoyl-ACP desaturase in developing rapeseed embryos during storage lipid biosynthesis. The resulting transgenic plants showed dramatically increased stearate levels in the seeds. A continuous distribution of stearate levels from 2% to 40% was observed in seeds of a transgenic B. napus plant, illustrating the potential to engineer specialized seed oil compositions.

  15. Lipase and esterase-catalyzed acylation of hetero-substituted nitrogen nucleophiles in water and organic solvents.

    PubMed

    Hacking, M A; Akkus, H; van Rantwijk, F; Sheldon, R A

    2000-04-01

    The lipase- and esterase-catalyzed acylations of hydroxylamine and hydrazine derivatives with octanoic acid and ethyl octanoate are described. The influence of solvent and nucleophile on the initial reaction rate was investigated for a number of free and immobilized enzymes. Initial rates were highest in water, but the overall productivity was optimal in dioxane. Octanoic acid (250 g/L) was converted for 93% into the hydroxamic acid in 36 h with only 1% (w/w) Candida antarctica lipase B (Novozym 435) in dioxane at 40 degrees C. This translates to a catalyst productivity of 68.5 g. g(-1). day(-1) and a space time yield of 149 g. L(-1). day(-1), unprecedented figures in the direct reaction of an acid with a nitrogen nucleophile in an organic solvent.

  16. Membrane Topology and Transient Acylation of Toxoplasma gondii Glycosylphosphatidylinositols

    PubMed Central

    Kimmel, Jürgen; Smith, Terry K.; Azzouz, Nahid; Gerold, Peter; Seeber, Frank; Lingelbach, Klaus; Dubremetz, Jean-François; Schwarz, Ralph T.

    2006-01-01

    Using hypotonically permeabilized Toxoplasma gondii tachyzoites, we investigated the topology of the free glycosylphosphatidylinositols (GPIs) within the endoplasmic reticulum (ER) membrane. The morphology and permeability of parasites were checked by electron microscopy and release of a cytosolic protein. The membrane integrity of organelles (ER and rhoptries) was checked by protease protection assays. In initial experiments, GPI biosynthetic intermediates were labeled with UDP-[6-3H]GlcNAc in permeabilized parasites, and the transmembrane distribution of the radiolabeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). A new early intermediate with an acyl modification on the inositol was identified, indicating that inositol acylation also occurs in T. gondii. A significant portion of the early GPI intermediates (GlcN-PI and GlcNAc-PI) could be hydrolyzed following PI-PLC treatment, indicating that these glycolipids are predominantly present in the cytoplasmic leaflet of the ER. Permeabilized T. gondii parasites labeled with either GDP-[2-3H]mannose or UDP-[6-3H]glucose showed that the more mannosylated and side chain (Glc-GalNAc)-modified GPI intermediates are also preferentially localized in the cytoplasmic leaflet of the ER. PMID:16896225

  17. Fatty acid hydrolysis of acyl marinobactin siderophores by Marinobacter acylases.

    PubMed

    Kem, Michelle P; Naka, Hiroaki; Iinishi, Akira; Haygood, Margo G; Butler, Alison

    2015-01-27

    The marine bacteria Marinobacter sp. DS40M6 and Marinobacter nanhaiticus D15-8W produce a suite of acyl peptidic marinobactin siderophores to acquire iron under iron-limiting conditions. During late-log phase growth, the marinobactins are hydrolyzed to form the marinobactin headgroup with release of the corresponding fatty acid tail. The bntA gene, a homologue of the Pseudomonas aeruginosa pyoverdine acylase gene, pvdQ, was identified from Marinobacter sp. DS40M6. A bntA knockout mutant of Marinobacter sp. DS40M6 produced the suite of acyl marinobactins A-E, without the usual formation of the marinobactin headgroup. Another marinobactin-producing species, M. nanhaiticus D15-8W, is predicted to have two pvdQ homologues, mhtA and mhtB. MhtA and MhtB have 67% identical amino acid sequences. MhtA catalyzes hydrolysis of the apo-marinobactin siderophores as well as the quorum sensing signaling molecule, dodecanoyl-homoserine lactone. In contrast to hydrolysis of the suite of apo-marinobactins by MhtA, hydrolysis of the iron(III)-bound marinobactins was not observed. PMID:25588131

  18. Site‐Selective Acylations with Tailor‐Made Catalysts

    PubMed Central

    Huber, Florian

    2016-01-01

    Abstract The acylation of alcohols catalyzed by N,N‐dimethylamino pyridine (DMAP) is, despite its widespread use, sometimes confronted with substrate‐specific problems: For example, target compounds with multiple hydroxy groups may show insufficient selectivity for one hydroxyl, and the resulting product mixtures are hardly separable. Here we describe a concept that aims at tailor‐made catalysts for the site‐specific acylation. To this end, we introduce a catalyst library where each entry is constructed by connecting a variable and readily tuned peptide scaffold with a catalytically active unit based on DMAP. For selected examples, we demonstrate how library screening leads to the identification of optimized catalysts, and the substrates of interest can be converted with a markedly enhanced site‐selectivity compared with only DMAP. Furthermore, substrate‐optimized catalysts of this type can be used to selectively convert “their” substrate in the presence of structurally similar compounds, an important requisite for reactions with mixtures of substances. PMID:26970553

  19. Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

    PubMed Central

    Vick, Jacob E.; Clomburg, James M.; Blankschien, Matthew D.; Chou, Alexander; Kim, Seohyoung

    2014-01-01

    We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012, http://dx.doi.org/10.1021/sb3000782). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a β-oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled ΔfabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in E. coli. PMID:25527535

  20. Molecular mechanism of a hotdog-fold acyl-CoA thioesterase.

    PubMed

    Cantu, David C; Ardèvol, Albert; Rovira, Carme; Reilly, Peter J

    2014-07-14

    Thioesterases are enzymes that hydrolyze thioester bonds between a carbonyl group and a sulfur atom. They catalyze key steps in fatty acid biosynthesis and metabolism, as well as polyketide biosynthesis. The reaction molecular mechanism of most hotdog-fold acyl-CoA thioesterases remains unknown, but several hypotheses have been put forward in structural and biochemical investigations. The reaction of a human thioesterase (hTHEM2), representing a thioesterase family with a hotdog fold where a coenzyme A moiety is cleaved, was simulated by quantum mechanics/molecular mechanics metadynamics techniques to elucidate atomic and electronic details of its mechanism, its transition-state conformation, and the free energy landscape of the process. A single-displacement acid-base-like mechanism, in which a nucleophilic water molecule is activated by an aspartate residue acting as a base, was found, confirming previous experimental proposals. The results provide unambiguous evidence of the formation of a tetrahedral-like transition state. They also explain the roles of other conserved active-site residues during the reaction, especially that of a nearby histidine/serine pair that protonates the thioester sulfur atom, the participation of which could not be elucidated from mutation analyses alone. PMID:24894958

  1. Enzymatic production of biodiesel from microalgal oil using ethyl acetate as an acyl acceptor.

    PubMed

    Alavijeh, Razieh Shafiee; Tabandeh, Fatemeh; Tavakoli, Omid; Karkhane, Aliasghar; Shariati, Parvin

    2015-01-01

    Microalgae have become an important source of biomass for biodiesel production. In enzymatic transesterification reaction, the enzyme activity is decreased in presence of alcohols. The use of different acyl acceptors such as methyl/ethyl acetate is suggested as an alternative and effective way to overcome this problem. In this study, ethyl acetate was used for the first time in the enzymatic production of biodiesel by using microalga, Chlorella vulgaris, as a triglyceride source. Enzymatic conversion of such fatty acids to biodiesel was catalyzed by Novozym 435 as an efficient immobilized lipase which is extensively used in biodiesel production. The best conversion yield of 66.71% was obtained at the ethyl acetate to oil molar ratio of 13:1 and Novozym 435 concentration of 40%, based on the amount of oil, and a time period of 72 h at 40℃. The results showed that ethyl acetate have no adverse effect on lipase activity and the biodiesel amount was not decreased even after seven transesterification cycles, so ethyl acetate has a great potential to be substituted for short-chain alcohols in transesterification reaction. PMID:25742923

  2. Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5.

    PubMed

    Brigidi, G Stefano; Santyr, Brendan; Shimell, Jordan; Jovellar, Blair; Bamji, Shernaz X

    2015-01-01

    Synaptic plasticity is mediated by the dynamic localization of proteins to and from synapses. This is controlled, in part, through activity-induced palmitoylation of synaptic proteins. Here we report that the ability of the palmitoyl-acyl transferase, DHHC5, to palmitoylate substrates in an activity-dependent manner is dependent on changes in its subcellular localization. Under basal conditions, DHHC5 is bound to PSD-95 and Fyn kinase, and is stabilized at the synaptic membrane through Fyn-mediated phosphorylation of a tyrosine residue within the endocytic motif of DHHC5. In contrast, DHHC5's substrate, δ-catenin, is highly localized to dendritic shafts, resulting in the segregation of the enzyme/substrate pair. Neuronal activity disrupts DHHC5/PSD-95/Fyn kinase complexes, enhancing DHHC5 endocytosis, its translocation to dendritic shafts and its association with δ-catenin. Following DHHC5-mediated palmitoylation of δ-catenin, DHHC5 and δ-catenin are trafficked together back into spines where δ-catenin increases cadherin stabilization and recruitment of AMPA receptors to the synaptic membrane. PMID:26334723

  3. Enzymatic production of biodiesel from microalgal oil using ethyl acetate as an acyl acceptor.

    PubMed

    Alavijeh, Razieh Shafiee; Tabandeh, Fatemeh; Tavakoli, Omid; Karkhane, Aliasghar; Shariati, Parvin

    2015-01-01

    Microalgae have become an important source of biomass for biodiesel production. In enzymatic transesterification reaction, the enzyme activity is decreased in presence of alcohols. The use of different acyl acceptors such as methyl/ethyl acetate is suggested as an alternative and effective way to overcome this problem. In this study, ethyl acetate was used for the first time in the enzymatic production of biodiesel by using microalga, Chlorella vulgaris, as a triglyceride source. Enzymatic conversion of such fatty acids to biodiesel was catalyzed by Novozym 435 as an efficient immobilized lipase which is extensively used in biodiesel production. The best conversion yield of 66.71% was obtained at the ethyl acetate to oil molar ratio of 13:1 and Novozym 435 concentration of 40%, based on the amount of oil, and a time period of 72 h at 40℃. The results showed that ethyl acetate have no adverse effect on lipase activity and the biodiesel amount was not decreased even after seven transesterification cycles, so ethyl acetate has a great potential to be substituted for short-chain alcohols in transesterification reaction.

  4. The fatty acyl-CoA reductase Waterproof mediates airway clearance in Drosophila.

    PubMed

    Jaspers, Martin H J; Pflanz, Ralf; Riedel, Dietmar; Kawelke, Steffen; Feussner, Ivo; Schuh, Reinhard

    2014-01-01

    The transition from a liquid to a gas filled tubular network is the prerequisite for normal function of vertebrate lungs and invertebrate tracheal systems. However, the mechanisms underlying the process of gas filling remain obscure. Here we show that waterproof, encoding a fatty acyl-CoA reductase (FAR), is essential for the gas filling of the tracheal tubes during Drosophila embryogenesis, and does not affect branch network formation or key tracheal maturation processes. However, electron microscopic analysis reveals that in waterproof mutant embryos the formation of the outermost tracheal cuticle sublayer, the envelope, is disrupted and the hydrophobic tracheal coating is damaged. Genetic and gain-of-function experiments indicate a non-cell-autonomous waterproof function for the beginning of the tracheal gas filling process. Interestingly, Waterproof reduces very long chain fatty acids of 24 and 26 carbon atoms to fatty alcohols. Thus, we propose that Waterproof plays a