Science.gov

Sample records for acetyl-coa carboxylase phosphorylation

  1. A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation

    PubMed Central

    Wei, Jia; Zhang, Yixiao; Yu, Tai-Yuan; Sadre-Bazzaz, Kianoush; Rudolph, Michael J; Amodeo, Gabriele A; Symington, Lorraine S; Walz, Thomas; Tong, Liang

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and attractive targets for drug discovery. Eukaryotic acetyl-CoA carboxylases are 250 kDa single-chain, multi-domain enzymes and function as dimers and higher oligomers. Their catalytic activity is tightly regulated by phosphorylation and other means. Here we show that yeast ACC is directly phosphorylated by the protein kinase SNF1 at residue Ser1157, which potently inhibits the enzyme. Crystal structure of three ACC central domains (AC3–AC5) shows that the phosphorylated Ser1157 is recognized by Arg1173, Arg1260, Tyr1113 and Ser1159. The R1173A/R1260A double mutant is insensitive to SNF1, confirming that this binding site is crucial for regulation. Electron microscopic studies reveal dramatic conformational changes in the holoenzyme upon phosphorylation, likely owing to the dissociation of the biotin carboxylase domain dimer. The observations support a unified molecular mechanism for the regulation of ACC by phosphorylation as well as by the natural product soraphen A, a potent inhibitor of eukaryotic ACC. These molecular insights enhance our understanding of acetyl-CoA carboxylase regulation and provide a basis for drug discovery. PMID:27990296

  2. Phosphorylation of chloroplast ribulose bisphosphate carboxylase/oxygenase small subunit by an envelope-bound protein kinase in situ.

    PubMed

    Soll, J; Buchanan, B B

    1983-06-10

    A new protein kinase of the cAMP independent type was found to be bound to the outer envelope membrane of spinach chloroplasts. While stimulated by Mg2+ and inhibited by ADP, the enzyme showed no response to conventional protein substrates and was essentially independent of pH in the physiological (pH 7 to 8) range. The new protein kinase phosphorylated the mature form of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase and, to a lesser extent, an unidentified 24-kDa polypeptide, both of which were bound to the outer envelope membrane. The results suggest that phosphorylation of cytoplasmically synthesized protein constituents of chloroplasts is involved in their transport through the chloroplast envelope membrane barrier.

  3. In vivo phosphorylation of phosphoenolpyruvate carboxylase in Egeria densa, a submersed aquatic species.

    PubMed

    Lara, M V; Casati, P; Andreo, C S

    2001-04-01

    In vivo phosphorylation of PEPC in Egeria densa was studied using plants at high temperature and in light, and plants kept at low temperature and in light. The isoform induced by high temperature and light was more phosphorylated in the light. Changes in kinetic and regulatory properties correlated with changes in the phosphorylation state of PEPC.

  4. The bacterial-type phosphoenolpyruvate carboxylase isozyme from developing castor oil seeds is subject to in vivo regulatory phosphorylation at serine-451.

    PubMed

    Dalziel, Katie J; O'Leary, Brendan; Brikis, Carolyne; Rao, Srinath K; She, Yi-Min; Cyr, Terry; Plaxton, William C

    2012-04-05

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate-metabolism. In developing castor oil seeds (COS) a novel allosterically-densensitized 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between 107-kDa plant-type PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. Mass spectrometry and immunoblotting with anti-phosphoSer451 specific antibodies established that COS BTPC is in vivo phosphorylated at Ser451, a highly conserved target residue that occurs within an intrinsically disordered region. This phosphorylation was enhanced during COS development or in response to depodding. Kinetic characterization of a phosphomimetic (S451D) mutant indicated that Ser451 phosphorylation inhibits the catalytic activity of BTPC subunits within the Class-2 PEPC complex.

  5. New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination

    PubMed Central

    Ruiz-Ballesta, Isabel; Baena, Guillermo; Gandullo, Jacinto; Wang, Liqun; She, Yi-Min; Plaxton, William Charles; Echevarría, Cristina

    2016-01-01

    Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV–V when coleoptiles initiate the formation of the photosynthetic tissues. PMID:27194739

  6. Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser425 provides a further tier of enzyme control in developing castor oil seeds.

    PubMed

    O'Leary, Brendan; Rao, Srinath K; Plaxton, William C

    2011-01-01

    PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate metabolism. Two distinct oligomeric PEPC classes were discovered in developing COS (castor oil seeds). Class-1 PEPC is a typical homotetramer of 107 kDa PTPC (plant-type PEPC) subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octamer arises from a tight interaction between Class-1 PEPC and 118 kDa BTPC (bacterial-type PEPC) subunits. Mass spectrometric analysis of immunopurified COS BTPC indicated that it is subject to in vivo proline-directed phosphorylation at Ser425. We show that immunoblots probed with phosphorylation site-specific antibodies demonstrated that Ser425 phosphorylation is promoted during COS development, becoming maximal at stage IX (maturation phase) or in response to depodding. Kinetic analyses of a recombinant, chimaeric Class-2 PEPC containing phosphomimetic BTPC mutant subunits (S425D) indicated that Ser425 phosphorylation results in significant BTPC inhibition by: (i) increasing its Km(PEP) 3-fold, (ii) reducing its I50 (L-malate and L-aspartate) values by 4.5- and 2.5-fold respectively, while (iii) decreasing its activity within the physiological pH range. The developmental pattern and kinetic influence of Ser425 BTPC phosphorylation is very distinct from the in vivo phosphorylation/activation of COS Class-1 PEPC's PTPC subunits at Ser11. Collectively, the results establish that BTPC's phospho-Ser425 content depends upon COS developmental and physiological status and that Ser425 phosphorylation attenuates the catalytic activity of BTPC subunits within a Class-2 PEPC complex. To the best of our knowledge, this study provides the first evidence for protein phosphorylation as a mechanism for the in vivo control of vascular plant BTPC activity.

  7. Coimmunopurification of phosphorylated bacterial- and plant-type phosphoenolpyruvate carboxylases with the plastidial pyruvate dehydrogenase complex from developing castor oil seeds.

    PubMed

    Uhrig, R Glen; O'Leary, Brendan; Spang, H Elizabeth; MacDonald, Justin A; She, Yi-Min; Plaxton, William C

    2008-03-01

    The phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS; Ricinus communis) endosperm was assessed using coimmunopurification (co-IP) followed by proteomic analysis. Earlier studies suggested that immunologically unrelated 107-kD plant-type PEPCs (p107/PTPC) and 118-kD bacterial-type PEPCs (p118/BTPC) are subunits of an unusual 910-kD hetero-octameric class 2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 because p118 quantitatively coimmunopurified with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PEPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation motif characteristic of PTPCs, Pro-Q Diamond phosphoprotein staining, immunoblotting with phospho-serine (Ser)/threonine Akt substrate IgG, and phosphate-affinity PAGE established that coimmunopurified p118 was multiphosphorylated at unique Ser and/or threonine residues. Tandem mass spectrometric analysis of an endoproteinase Lys-C p118 peptide digest demonstrated that Ser-425 is subject to in vivo proline-directed phosphorylation. The co-IP of p118 with p107 did not appear to be influenced by their phosphorylation status. Because p118 phosphorylation was unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118's in vivo phosphorylation. A 110-kD PTPC coimmunopurified with p118 and p107 when depodded COS was used. The plastidial pyruvate dehydrogenase complex (PDC(pl)) was identified as a novel PEPC interactor. Thus, a putative metabolon involving PEPC and PDC(pl) could function to channel carbon from phosphoenolpyruvate to acetyl-coenzyme A and/or to recycle CO(2) from PDC(pl) to PEPC.

  8. Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea1[W][OA

    PubMed Central

    Shane, Michael W.; Fedosejevs, Eric T.; Plaxton, William C.

    2013-01-01

    Accumulating evidence indicates important functions for phosphoenolpyruvate (PEP) carboxylase (PEPC) in inorganic phosphate (Pi)-starved plants. This includes controlling the production of organic acid anions (malate, citrate) that are excreted in copious amounts by proteoid roots of nonmycorrhizal species such as harsh hakea (Hakea prostrata). This, in turn, enhances the bioavailability of mineral-bound Pi by solubilizing Al3+, Fe3+, and Ca2+ phosphates in the rhizosphere. Harsh hakea thrives in the nutrient-impoverished, ancient soils of southwestern Australia. Proteoid roots from Pi-starved harsh hakea were analyzed over 20 d of development to correlate changes in malate and citrate exudation with PEPC activity, posttranslational modifications (inhibitory monoubiquitination versus activatory phosphorylation), and kinetic/allosteric properties. Immature proteoid roots contained an equivalent ratio of monoubiquitinated 110-kD and phosphorylated 107-kD PEPC polypeptides (p110 and p107, respectively). PEPC purification, immunoblotting, and mass spectrometry indicated that p110 and p107 are subunits of a 430-kD heterotetramer and that they both originate from the same plant-type PEPC gene. Incubation with a deubiquitinating enzyme converted the p110:p107 PEPC heterotetramer of immature proteoid roots into a p107 homotetramer while significantly increasing the enzyme’s activity under suboptimal but physiologically relevant assay conditions. Proteoid root maturation was paralleled by PEPC activation (e.g. reduced Km [PEP] coupled with elevated I50 [malate and Asp] values) via in vivo deubiquitination of p110 to p107, and subsequent phosphorylation of the deubiquitinated subunits. This novel mechanism of posttranslational control is hypothesized to contribute to the massive synthesis and excretion of organic acid anions that dominates the carbon metabolism of the mature proteoid roots. PMID:23407057

  9. Re-examination of the roles of PEP and Mg2+ in the reaction catalysed by the phosphorylated and non-phosphorylated forms of phosphoenolpyruvate carboxylase from leaves of Zea mays. Effects of the activators glucose 6-phosphate and glycine.

    PubMed Central

    Tovar-Méndez, A; Rodríguez-Sotres, R; López-Valentín, D M; Muñoz-Clares, R A

    1998-01-01

    To study the effects of phosphoenolpyruvate (PEP) and Mg2+ on the activity of the non-phosphorylated and phosphorylated forms of phosphoenolpyruvate carboxylase (PEPC) from Zea mays leaves, steady-state measurements have been carried out with the free forms of PEP (fPEP) and Mg2+ (fMg2+), both in a near-physiological concentration range. At pH 7.3, in the absence of activators, the initial velocity data obtained with both forms of the enzyme are consistent with the exclusive binding of MgPEP to the active site and of fPEP to an activating allosteric site. At pH 8.3, and in the presence of saturating concentrations of glucose 6-phosphate (Glc6P) or Gly, the free species also combined with the active site in the free enzyme, but with dissociation constants at least 35-fold that estimated for MgPEP. The latter dissociation constant was lowered to the same extent by saturating Glc6P and Gly, to approx. one-tenth and one-sixteenth in the non-phosphorylated and phosphorylated enzymes respectively. When Glc6P is present, fPEP binds to the active site in the free enzyme better than fMg2+, whereas the metal ion binds better in the presence of Gly. Saturation of the enzyme with Glc6P abolished the activation by fPEP, consistent with a common binding site, whereas saturation with Gly increased the affinity of the allosteric site for fPEP. Under all the conditions tested, our results suggest that fPEP is not able to combine with the allosteric site in the free enzyme, i.e. it cannot combine until after MgPEP, fPEP or fMg2+ are bound at the active site. The physiological role of Mg2+ in the regulation of the enzyme is only that of a substrate, mainly as part of the MgPEP complex. The kinetic properties of maize leaf PEPC reported here are consistent with the enzyme being well below saturation under the physiological concentrations of fMg2+ and PEP, particularly during the dark period; it is therefore suggested that the basal PEPC activity in vivo is very low, but highly

  10. Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase by a Ca2+-dependent protein kinase suggests a link between Ca2+ signalling and anaplerotic pathway control in developing castor oil seeds.

    PubMed

    Hill, Allyson T; Ying, Sheng; Plaxton, William C

    2014-02-15

    The aim of the present study was to characterize the native protein kinase [BTPC (bacterial-type phosphoenolpyruvate carboxylase)-K (BTPC Ser451 kinase)] that in vivo phosphorylates Ser451 of the BTPC subunits of an unusual Class-2 PEP (phosphoenolpyruvate) carboxylase hetero-octameric complex of developing COS (castor oil seeds). COS BTPC-K was highly purified by PEG fractionation and hydrophobic size-exclusion anion-exchange and affinity chromatographies. BTPC-K phosphorylated BTPC strictly at Ser451 (Km=1.0 μM; pH optimum=7.3), a conserved target residue occurring within an intrinsically disordered region, as well as the protein histone III-S (Km=1.7 μM), but not a COS plant-type PEP carboxylase or sucrose synthase or α-casein. Its activity was Ca2+- (K0.5=2.7 μM) and ATP- (Km=6.6 μM) dependent, and markedly inhibited by trifluoperazine, 3-phosphoglycerate and PEP, but insensitive to calmodulin or 14-3-3 proteins. BTPC-K exhibited a native molecular mass of ~63 kDa and was soluble rather than membrane-bound. Inactivation and reactivation occurred upon BTPC-K's incubation with GSSG and then DTT respectively. Ser451 phosphorylation by BTPC-K inhibited BTPC activity by ~50% when assayed under suboptimal conditions (pH 7.3, 1 mM PEP and 10 mM L-malate). Our collective results indicate a possible link between cytosolic Ca2+ signalling and anaplerotic flux control in developing COS.

  11. Plant, cell, and molecular mechanisms of abscisic-acid regulation of stomatal apertures. In vivo phosphorylation of phosphoenolpyruvate carboxylase in guard cells of Vicia faba L. is enhanced by fusicoccin and suppressed by abscisic acid

    SciTech Connect

    Du, Z.; Aghoram, K.; Outlaw, W.H. Jr.

    1996-12-31

    Plants regulate water loss and CO{sub 2} gain by modulating the aperture sizes of stomata that penetrate the epidermis. Aperture size itself is increased by osmolyte accumulation and consequent turgor increase in the pair of guard cells that flank each stoma. Guard-cell phosphoenolpyruvate carboxylase, which catalyzes the regulated step leading to malate synthesis, is crucial for charge and pH maintenance during osmolyte accumulation. Regulation of this cytosolic enzyme by effectors is well documented, but additional regulation by posttranslational modification is predicted by the alteration of PEPC kinetics during stomatal opening. In this study, the authors have investigated whether this alteration is associated with the phosphorylation status of this enzyme. Using sonicated epidermal peels (isolated guard cells) pre-loaded with {sub 32}PO{sub 4}, the authors induced stomatal opening and guard-cell malate accumulation by incubation with 5 {micro}M fusicoccin (FC). In corroboratory experiments, guard cells were incubated with 5 {micro}M fusicoccin (FC). In corroboratory experiments, guard cells were incubated with the FC antagonist, 10 {micro}M abscisic acid (ABA). The phosphorylation status of PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. PEPC was phosphorylated when stomata were stimulated to open, and phosphorylation was lessened by incubation with ABA.

  12. Inhibitors of Pyruvate Carboxylase

    PubMed Central

    Zeczycki, Tonya N.; Maurice, Martin St.; Attwood, Paul V.

    2010-01-01

    This review aims to discuss the varied types of inhibitors of biotin-dependent carboxylases, with an emphasis on the inhibitors of pyruvate carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of pyruvate carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin – biotin, FTP – ATP binding site, oxamate – pyruvate binding site, phosphonoacetate – binding site of the putative carboxyphosphate intermediate. PMID:22180764

  13. Prebiotic Fiber Increases Hepatic Acetyl CoA Carboxylase Phosphorylation and Suppresses Glucose-Dependent Insulinotropic Polypeptide Secretion More Effectively When Used with Metformin in Obese Rats1,2

    PubMed Central

    Pyra, Kim A.; Saha, Dolan C.; Reimer, Raylene A.

    2013-01-01

    Independently, metformin (MET) and the prebiotic, oligofructose (OFS), have been shown to increase glucagon-like peptide (GLP-1) secretion. Our objective was to determine whether using OFS as an adjunct with MET augments GLP-1 secretion in obese rats. Male, diet-induced obese Sprague Dawley rats were randomized to: 1) high-fat/-sucrose diet [HFHS; control (C); 20% fat, 50% sucrose wt:wt]; 2) HFHS+10% OFS (OFS); 3) HFHS + MET [300 mg/kg/d (MET)]; 4) HFHS+10% OFS+MET (OFS +MET). Body composition, glycemia, satiety hormones, and mechanisms related to dipeptidyl peptidase 4 (DPP4) activity in plasma, hepatic AMP-activated protein kinase (AMPK; Western blots), and gut microbiota (qPCR) were examined. Direct effects of MET and SCFA were examined in human enteroendocrine cells. The interaction between OFS and MET affected fat mass, hepatic TG, secretion of glucose-dependent insulinotropic polypeptide (GIP) and leptin, and AMPKα2 mRNA and phosphorylated acetyl CoA carboxylase (pACC) levels (P < 0.05). Combined, OFS and MET reduced GIP secretion to a greater extent than either treatment alone (P < 0.05). The hepatic pACC level was increased by OFS+MET by at least 50% above all other treatments, which did not differ from each other (P < 0.05). OFS decreased plasma DPP4 activity (P < 0.001). Cecal Bifidobacteria (P < 0.001) were markedly increased and C. leptum decreased (P < 0.001) with OFS consumption. In human enteroendocrine cells, the interaction between MET and SCFA affected GLP-1 secretion (P < 0.04) but was not associated with higher GLP-1 than the highest individual doses. In conclusion, the combined actions of OFS and MET were associated with important interaction effects that have the potential to improve metabolic outcomes associated with obesity. PMID:22223580

  14. The dynamic organization of fungal acetyl-CoA carboxylase

    PubMed Central

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. PMID:27073141

  15. The dynamic organization of fungal acetyl-CoA carboxylase

    NASA Astrophysics Data System (ADS)

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-04-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

  16. Isolation, identification, and synthesis of 2-carboxyarabinitol 1-phosphate, a diurnal regulator of ribulase-bisphosphate carboxylase activity

    SciTech Connect

    Berry, J.A.; Lorimer, G.H.; Pierce, J.; Seemann, J.R.; Meek, J.; Freas, S.

    1987-02-01

    The diurnal change in activity of ribulose 1,5-bisphosphate (Rbu-1,5-P/sub 2/) carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39) of leaves of Phaseolus vulgaris is regulated (in part) by mechanisms that control the level of an endogenous inhibitor that binds tightly to the activated (carbamoylated) form of Rbu-1,5-P/sub 2/ carboxylase. This inhibitor was extracted from leaves and copurified with the Rbu-1,5-P/sub 2/ carboxylase of the leaves. Further purification by ion-exchange chromatography, adsorption to purified Rbu-1,5-P/sub 2/ carboxylase, barium precipitation, and HPLC separation yielded a phosphorylated compound that was a strong inhibitor of Rbu-1,5-P/sub 2/ carboxylase. The compound was analyzed by GC/MS, /sup 13/C NMR, and /sup 1/H NMR and shown to be 2-carboxyarabinitol 1-phosphate ((2-C-phosphohydroxymethyl)-D-ribonic acid). The structure of the isolated compound differs from the Rbu-1,5-P/sub 2/ carboxylase transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate only by the lack of the C-5 phosphate group. This difference results in a higher binding constant for the monophosphate compared with the bisphosphate. The less tightly bound compound acts in a light-dependent, reversible regulation of Rbu-1,5-P/sub 2/ carboxylase activity in vivo.

  17. Assay of ribulose bisphosphate carboxylase

    SciTech Connect

    Pike, C.; Berry, J.

    1987-04-01

    Assays of ribulose bisphosphate carboxylase (rubisco) can be used to illustrate many properties of photosynthetic systems. Many different leaves have been assayed with this standard procedure. The tissue is ground with a mortar and pestle in extraction buffer. The supernatant after centrifugation is used as the source of enzyme. Buffer, RuBP, (/sup 14/C)-NaHCO/sub 3/, and enzyme are combined in a scintillation vial; the reaction is run for 1 min at 30/sup 0/. The acid-stable products are counted. Reproducibility in student experiments has been excellent. The assay data can be combined with analyses of leaf properties such as fresh and dry weight, chlorophyll and protein content, etc. Students have done projects such as the response of enzyme to temperature and to various inhibitors. They also report on the use of a transition state analog, carboxyarabinitol bisphosphate, to titrate the molar concentration of rubisco molecules (active sites) in an enzyme sample. Thus, using crude extracts the catalytic activity of a sample can be compared to the absolute quantity of enzyme or to the turnover number.

  18. Protein phosphorylation and photorespiration.

    PubMed

    Hodges, M; Jossier, M; Boex-Fontvieille, E; Tcherkez, G

    2013-07-01

    Photorespiration allows the recycling of carbon atoms of 2-phosphoglycolate produced by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, proteins undergo a number of different post-translational modifications that include acetylation, methylation and ubiquitylation, but protein phosphorylation is probably the most common. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function, such as activity, subcellular localisation, capacity to interact with other proteins and stability. Recent data indicate that many photorespiratory enzymes can be phosphorylated, and thus it seems that the photorespiratory cycle is, in part, regulated by protein phosphorylation. In this review, the known phosphorylation sites of each Arabidopsis thaliana photorespiratory enzyme and several photorespiratory-associated proteins are described and discussed. A brief account of phosphoproteomic protocols is also given since the published data compiled in this review are the fruit of this approach.

  19. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  20. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

  1. Evolutionary history and biotechnological future of carboxylases.

    PubMed

    Schada von Borzyskowski, Lennart; Rosenthal, Raoul G; Erb, Tobias J

    2013-11-01

    Carbon dioxide (CO2) is a potent greenhouse gas whose presence in the atmosphere is a critical factor for global warming. At the same time atmospheric CO2 is also a cheap and readily available carbon source that can in principle be used to synthesize value-added products. However, as uncatalyzed chemical CO2-fixation reactions usually require quite harsh conditions to functionalize the CO2 molecule, not many processes have been developed that make use of CO2. In contrast to synthetical chemistry, Nature provides a multitude of different carboxylating enzymes whose carboxylating principle(s) might be exploited in biotechnology. This review focuses on the biochemical features of carboxylases, highlights possible evolutionary scenarios for the emergence of their reactivity, and discusses current, as well as potential future applications of carboxylases in organic synthesis, biotechnology and synthetic biology.

  2. Studies of vitamin K-dependent carboxylase

    SciTech Connect

    Wood, G.M.

    1986-01-01

    Carboxylase was studied in detergent solubilized rat liver microsomes, using the peptide substrate Phe-Leu-(..gamma..-/sup 3/H)-Glu-Glu-Leu. Cleavage of the ..gamma..-C-H bond in Glu was measured as the release of /sup 3/H from this peptide to water, carboxylation was measured as the incorporation of H/sup 14/CO/sub 3/-into the peptide, and KO formation was measured by an HPLC assay. All three products could be measured simultaneously, and this system was used to examine the effects of cyanide, manganese, tetrachloropyridinol, and Boc-SerP-SerP-Leu-OMe on the separate steps of the carboxylase reaction. Vitamin K-epoxide formation was studied separately from the other reactions, and it was found that in the absence of a Glu-containing substrate, carboxylase catalyzed the uncoupled formation of KO from KH/sub 2/ and O/sub 2/. The stoichiometry of product formation (GLa, KO, and ..gamma..-protons) was measured, and the results obtained were all in agreement with the values predicted from the proposed mechanism. When all of the substrates were saturating, the stoichiometry of ..gamma..-C-H bond cleavage, carboxylation, and KO formation was 1:1:1.

  3. Structure and function of biotin-dependent carboxylases

    PubMed Central

    Tong, Liang

    2012-01-01

    Biotin-dependent carboxylases include acetyl-CoA carboxylase (ACC), propionyl-CoA carboxylase (PCC), 3-methylcrotonyl-CoA carboxylase (MCC), geranyl-CoA carboxylase (GCC), pyruvate carboxylase (PC), and urea carboxylase (UC). They contain biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) components. These enzymes are widely distributed in nature and have important functions in fatty acid metabolism, amino acid metabolism, carbohydrate metabolism, polyketide biosynthesis, urea utilization, and other cellular processes. ACCs are also attractive targets for drug discovery against type 2 diabetes, obesity, cancer, microbial infections, and other diseases, and the plastid ACC of grasses is the target of action of three classes of commercial herbicides. Deficiencies in the activities of PCC, MCC or PC are linked to serious diseases in humans. Our understanding of these enzymes has been greatly enhanced over the past few years by the crystal structures of the holoenzymes of PCC, MCC, PC, and UC. The structures reveal unanticipated features in the architectures of the holoenzymes, including the presence of previously unrecognized domains, and provide a molecular basis for understanding their catalytic mechanism as well as the large collection of disease-causing mutations in PCC, MCC and PC. This review will summarize the recent advances in our knowledge on the structure and function of these important metabolic enzymes. PMID:22869039

  4. Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli†

    PubMed Central

    Zeczycki, Tonya N.; Maurice, Martin St.; Jitrapakdee, Sarawut; Wallace, John C.; Attwood, Paul V.; Cleland, W. Wallace

    2009-01-01

    The effects of mutations in the active site of the carboxyl transferase domain of R. etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of MgADP by carbamoyl phosphate and the bicarbonate-dependent ATPase reaction. Additional studies with these mutants examined the effect of pyruvate and oxamate on the reactions of the biotin carboxylase domain. From these mutagenic studies, putative roles for catalytically relevant active site residues were assigned and a more accurate description of the mechanism of the carboxyl transferase domain is presented. The T882A mutant showed no catalytic activity for reactions involving the carboxyl transferase domain, but surprisingly showed a 7- and 3.5-fold increase in activity, as compared to the wild-type enzyme, for the ADP phosphorylation and bicarbonate-dependent ATPase reactions, respectively. Furthermore, the partial inhibition of the T882A catalyzed BC domain reactions by oxamate and pyruvate further supports the critical role of Thr882 in the proton transfer between biotin and pyruvate in the carboxyl transferase domain. The catalytic mechanism appears to involve the decarboxylation of carboxybiotin and proton removal from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882. The resulting enolpyruvate then reacts with CO2 to form oxaloacetate and complete the reaction. PMID:19341298

  5. Novel Insights into the Biotin Carboxylase Domain Reactions of Pyruvate Carboxylase from Rhizobium etli†

    PubMed Central

    Zeczycki, Tonya N.; Menefee, Ann L.; Adina-Zada, Abdussalam; Jitrapakdee, Sarawut; Surinya, Kathy H.; Wallace, John C.; Attwood, Paul V.; St. Maurice, Martin; Cleland, W. Wallace

    2011-01-01

    The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO3− deprotonation (Glu305 and Arg301) and biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in kcat for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO3−, Lys245, Glu218 and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the biotin carboxylase and carboxyl transferase domains were uncoupled in the R353M-catalyzed reactions, indicating that Arg353 may not only facilitate the formation of the biotin enolate, but also assist in coordinating catalysis between the two spatially distinct active sites. The 2.5 and 4-fold increase in kcat for the full reverse reaction with the R353K and R353M mutants, respectively, suggests that mutation of Arg353 allows carboxybiotin increased access to the biotin carboxylase domain active site. The proposed chemical mechanism is initiated by the deprotonation of HCO3− by Glu305 and concurrent nucleophilic attack on the γ-phosphate of MgATP. The trianionic carboxyphosphate intermediate formed reversibly decomposes in the active site to CO2 and PO43−. PO43− then acts as the base to deprotonate the tethered biotin at the N1-position. Stabilized by interactions between the ureido oxygen and Arg353, the biotin-enolate reacts with CO2 to give carboxybiotin. The formation of a distinct salt

  6. Molecular evolution of urea amidolyase and urea carboxylase in fungi

    PubMed Central

    2011-01-01

    Background Urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. Urea amidolyase has been found only in some fungal species among eukaryotes. It contains two major domains: the amidase and urea carboxylase domains. A shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. Eukaryotic urea carboxylase has been found only in several fungal species and green algae. In order to elucidate the evolutionary origin of urea amidolyase and urea carboxylase, we studied the distribution of urea amidolyase, urea carboxylase, as well as other proteins including urease, across kingdoms. Results Among the 64 fungal species we examined, only those in two Ascomycota classes (Sordariomycetes and Saccharomycetes) had the urea amidolyase sequences. Urea carboxylase was found in many but not all of the species in the phylum Basidiomycota and in the subphylum Pezizomycotina (phylum Ascomycota). It was completely absent from the class Saccharomycetes (phylum Ascomycota; subphylum Saccharomycotina). Four Sordariomycetes species we examined had both the urea carboxylase and the urea amidolyase sequences. Phylogenetic analysis showed that these two enzymes appeared to have gone through independent evolution since their bacterial origin. The amidase domain and the urea carboxylase domain sequences from fungal urea amidolyases clustered strongly together with the amidase and urea carboxylase sequences, respectively, from a small number of beta- and gammaproteobacteria. On the other hand, fungal urea carboxylase proteins clustered together with another copy of urea carboxylases distributed broadly among bacteria. The urease proteins were found in all the fungal species examined except for those of the subphylum Saccharomycotina. Conclusions We conclude that the urea amidolyase genes currently found only in fungi are the results of a horizontal gene transfer event from

  7. Insulin stimulates the dephosphorylation and activation of acetyl-CoA carboxylase.

    PubMed Central

    Witters, L A; Watts, T D; Daniels, D L; Evans, J L

    1988-01-01

    The mechanism underlying the ability of insulin to acutely activate acetyl-CoA carboxylase [acetyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.2; AcCoA-Case] has been examined in Fao Reuber hepatoma cells. Insulin promotes the rapid activation of AcCoACase, as measured in cell lysates, and this stimulation persists to the same degree after isolation of AcCoACase by avidin-Sepharose chromatography. The insulin-stimulated enzyme, as compared with control enzyme, exhibits an increase in both citrate-independent and -dependent activity and a decrease in the Ka for citrate. Direct examination of the phosphorylation state of isolated 32P-labeled AcCoACase after insulin exposure reveals a marked decrease in total enzyme phosphorylation coincident with activation. The dephosphorylation due to insulin appears to be restricted to the phosphorylation sites previously shown to regulate AcCoACase activity. All of these effects of insulin are mimicked by a low molecular weight autocrine factor, tentatively identified as an oligosaccharide, present in conditioned medium of hepatoma cells. These data suggest that insulin may activate AcCoACase by inhibiting the activity of protein kinase(s) or stimulating the activity of protein phosphatase(s) that control the phosphorylation state of the enzyme. Images PMID:2899891

  8. Ribulose 1,5-bisphosphate carboxylase and phosphoribulokinase in Prochloron

    NASA Technical Reports Server (NTRS)

    Berhow, M. A.; Mcfadden, B. A.

    1983-01-01

    Ribulose 1,5-bisphosphate (RuBP) carboxylase and phosphoribulokinase, enzymes in the reductive pentose-phosphate cycle, were measured in cell-free extracts of Prochloran didemni. The partial purification and characterization of RuBP carboxylase were described. Prochloron RuBP carboxylase, when purified by isopycnic centrifugation in reoriented linear 0.2 to 0.8 M sucrose gradients, sedimented to a position which corresponded to that of the 520,000-dalton spinach enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the Prochloron enzyme was composed of large and small subunits (MW = 57,500 and 18,800). Though results established that the enzymes RuBP carboxylase and phosphoribulokinase were present in levels comparable to other CO2-fixing microorganisms, it was suggested that other enzymes in the Calvin cycle limit growth or that additional enzymic insufficiencies exist.

  9. Evolution of the Phosphoenolpyruvate Carboxylase Protein Kinase Family in C3 and C4 Flaveria spp.1[W][OPEN

    PubMed Central

    Aldous, Sophia H.; Weise, Sean E.; Sharkey, Thomas D.; Waldera-Lupa, Daniel M.; Stühler, Kai; Mallmann, Julia; Groth, Georg; Gowik, Udo; Westhoff, Peter; Arsova, Borjana

    2014-01-01

    The key enzyme for C4 photosynthesis, Phosphoenolpyruvate Carboxylase (PEPC), evolved from nonphotosynthetic PEPC found in C3 ancestors. In all plants, PEPC is phosphorylated by Phosphoenolpyruvate Carboxylase Protein Kinase (PPCK). However, differences in the phosphorylation pattern exist among plants with these photosynthetic types, and it is still not clear if they are due to interspecies differences or depend on photosynthetic type. The genus Flaveria contains closely related C3, C3-C4 intermediate, and C4 species, which are evolutionarily young and thus well suited for comparative analysis. To characterize the evolutionary differences in PPCK between plants with C3 and C4 photosynthesis, transcriptome libraries from nine Flaveria spp. were used, and a two-member PPCK family (PPCKA and PPCKB) was identified. Sequence analysis identified a number of C3- and C4-specific residues with various occurrences in the intermediates. Quantitative analysis of transcriptome data revealed that PPCKA and PPCKB exhibit inverse diel expression patterns and that C3 and C4 Flaveria spp. differ in the expression levels of these genes. PPCKA has maximal expression levels during the day, whereas PPCKB has maximal expression during the night. Phosphorylation patterns of PEPC varied among C3 and C4 Flaveria spp. too, with PEPC from the C4 species being predominantly phosphorylated throughout the day, while in the C3 species the phosphorylation level was maintained during the entire 24 h. Since C4 Flaveria spp. evolved from C3 ancestors, this work links the evolutionary changes in sequence, PPCK expression, and phosphorylation pattern to an evolutionary phase shift of kinase activity from a C3 to a C4 mode. PMID:24850859

  10. Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

    PubMed Central

    Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

    1995-01-01

    Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves. PMID:7610168

  11. Characterization of ribulose diphosphate carboxylase and phosphoribulokinase from Thiobacillus thioparus and Thiobacillus neapolitanus.

    NASA Technical Reports Server (NTRS)

    Johnson, E. J.; Johnson, M. K.; Macelroy, R. D.

    1968-01-01

    Ribulose diphosphate carboxylase and phosphoribulokinase activity in chemosynthetic autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus, noting sedimentation and gel filtration characteristics

  12. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation.

    PubMed

    Ferreira, R M; Franco, E; Teixeira, A R

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (< 1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose bisphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues.

  13. Properties of ribulose diphosphate carboxylase immobilized on porous glass

    NASA Technical Reports Server (NTRS)

    Shapira, J.; Hanson, C. L.; Lyding, J. M.; Reilly, P. J.

    1974-01-01

    Ribulose-1,5-diphosphate carboxylase from spinach has been bound to arylamine porous glass with a diazo linkage and to alklamine porous glass with glutaraldehyde. Stability at elevated temperatures and responses to changes of pH and ribulose-1,5-diphosphate, Mg(2+), and dithiothreitol concentrations were not significantly different from the soluble enzyme, though stability at 4 C was somewhat improved.

  14. Ribulose diphosphate carboxylase of the cyanobacterium Spirulina platensis

    SciTech Connect

    Terekhova, I.V.; Chernyad'ev, I.I.; Doman, N.G.

    1986-11-20

    The ribulose diphosphate (RDP) carboxylase activity of the cyanobacterium Spirulina platensis is represented by two peaks when a cell homogenate is centrifuged in a sucrose density gradient. In the case of differential centrifugation (40,000 g, 1 h), the activity of the enzyme was distributed between the supernatant liquid (soluble form) and the precipitate (carboxysomal form). From the soluble fraction, in which 80-95% of the total activity of the enzyme is concentrated, electrophoretically homogeneous RDP carboxylase was isolated by precipitation with ammonium sulfate and centrifugation in a sucrose density gradient. The purified enzyme possessed greater electrophoretic mobility in comparison with the RDP carboxylase of beans Vicia faba. The molecular weight of the enzyme, determined by gel filtration, was 450,000. The enzyme consists of monotypic subunits with a molecular weight of 53,000. The small subunits were not detected in electrophoresis in polyacrylamide gel in the presence of SDS after fixation and staining of the gels by various methods.

  15. Dimerization of the Bacterial Biotin Carboxylase Subunit Is Required for Acetyl Coenzyme A Carboxylase Activity In Vivo

    PubMed Central

    Smith, Alexander C.

    2012-01-01

    Acetyl coenzyme A (acteyl-CoA) carboxylase (ACC) is the first committed enzyme of the fatty acid synthesis pathway. Escherichia coli ACC is composed of four different proteins. The first enzymatic activity of the ACC complex, biotin carboxylase (BC), catalyzes the carboxylation of the protein-bound biotin moiety of another subunit with bicarbonate in an ATP-dependent reaction. Although BC is found as a dimer in cell extracts and the carboxylase activities of the two subunits of the dimer are interdependent, mutant BC proteins deficient in dimerization are reported to retain appreciable activity in vitro (Y. Shen, C. Y. Chou, G. G. Chang, and L. Tong, Mol. Cell 22:807–818, 2006). However, in vivo BC must interact with the other proteins of the complex, and thus studies of the isolated BC may not reflect the intracellular function of the enzyme. We have tested the abilities of three BC mutant proteins deficient in dimerization to support growth and report that the two BC proteins most deficient in dimerization fail to support growth unless expressed at high levels. In contrast, the wild-type protein supports growth at low expression levels. We conclude that BC must be dimeric to fulfill its physiological function. PMID:22037404

  16. An 'in situ' perfusion system suitable for investigating mammary-tissue metabolism in the lactating rat. Hormonal regulation of acetyl-CoA carboxylase.

    PubMed Central

    Clegg, R A; Calvert, D T

    1988-01-01

    A technique is described for the non-recirculating perfusion of inguinal/abdominal mammary tissue in situ in anaesthetized lactating rats. Tissue viability was maintained, without resort to infusion of vasoactive chemicals which may also be effectors of cellular metabolism, for at least 90 min. Total tissue adenine nucleotides (per mg of DNA) were somewhat decreased in perfused relative to non-perfused mammary tissue. DNA content (per g wet wt. of tissue) was diminished after 90 min of perfusion to approx. 65% of its value in control tissue. Adenylate energy-charge ratios were lower in perfused tissue in the absence of hormones than in control tissue. They were increased to control values by the presence of either insulin or isoprenaline in the perfusate. No changes occurred in flow rate of the perfusate that might account for these increases. In mammary tissue perfused without addition of hormones, acetyl-CoA carboxylase activities were similar to those measured in control tissue samples, although activity-ratio measurements implied some increase in the phosphorylation of this enzyme. Insulin or isoprenaline increased the activity of acetyl-CoA carboxylase, especially when this was measured at low concentrations of citrate. Confirming conclusions from previous experiments with mammary acini and explant preparations, insulin activated acetyl-CoA carboxylase in mammary tissue, but inhibition of its activity was not mediated by cyclic AMP. PMID:2895636

  17. Catalytic by-product formation and ligand binding by ribulose bisphosphate carboxylases from different phylogenies.

    PubMed

    Pearce, F Grant

    2006-11-01

    During catalysis, all Rubisco (D-ribulose-1,5-bisphosphate carboxylase/oxygenase) enzymes produce traces of several by-products. Some of these by-products are released slowly from the active site of Rubisco from higher plants, thus progressively inhibiting turnover. Prompted by observations that Form I Rubisco enzymes from cyanobacteria and red algae, and the Form II Rubisco enzyme from bacteria, do not show inhibition over time, the production and binding of catalytic by-products was measured to ascertain the underlying differences. In the present study we show that the Form IB Rubisco from the cyanobacterium Synechococcus PCC6301, the Form ID enzyme from the red alga Galdieria sulfuraria and the low-specificity Form II type from the bacterium Rhodospirillum rubrum all catalyse formation of by-products to varying degrees; however, the by-products are not inhibitory under substrate-saturated conditions. Study of the binding and release of phosphorylated analogues of the substrate or reaction intermediates revealed diverse strategies for avoiding inhibition. Rubisco from Synechococcus and R. rubrum have an increased rate of inhibitor release. G. sulfuraria Rubisco releases inhibitors very slowly, but has an increased binding constant and maintains the enzyme in an activated state. These strategies may provide information about enzyme dynamics, and the degree of enzyme flexibility. Our observations also illustrate the phylogenetic diversity of mechanisms for regulating Rubisco and raise questions about whether an activase-like mechanism should be expected outside the green-algal/higher-plant lineage.

  18. Phosphoenolpyruvate carboxylase in mistletoe leaves: Regulation of gene expression, protein content, and covalent modification.

    PubMed

    Li, Xuemei; Wanek, Wolfgang; Nehls, U.; Popp, Marianne; Hampp, Rüdiger; Rennenberg, Heinz; Einig, Werner

    2001-07-01

    Seasonal changes in the activity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31), a key enzyme in the interaction of carbohydrate and nitrogen metabolism, were studied in leaves of the C3 semiparasitic mistletoe, Viscum album, growing on different host trees. Maximum extractable PEPCase activities were higher in leaves of mistletoes growing on Betula pendula and Alnus glutinosa hosts compared with those on the conifers, Abies alba and Larix decidua. Independent of host, maximum extractable PEPCase activities were high in spring and autumn while low in summer. Samples with higher PEPCase activities showed higher amounts of PEPCase protein and higher PEPCase mRNA levels. A curvilinear correlation between leaf total nitrogen content and the maximum extractable PEPCase activity as well as PEPCase mRNA level suggested that nitrogen might affect the activity of PEPCase of mistletoe by up-regulating gene expression. In addition to extractable activity, seasonal changes of the PEPCase activation state, the ratio of activities resulting from limited:non-limited assays, were found, which was correlated to the variation of malate content in leaves of mistletoe. ATP-dependent activation of PEPCase was characterized by an increase in I0.5(L-malate), indicating that PEPCase of leaves of mistletoes is probably regulated via phosphorylation.

  19. Piperazine oxadiazole inhibitors of acetyl-CoA carboxylase.

    PubMed

    Bourbeau, Matthew P; Siegmund, Aaron; Allen, John G; Shu, Hong; Fotsch, Christopher; Bartberger, Michael D; Kim, Ki-Won; Komorowski, Renee; Graham, Melissa; Busby, James; Wang, Minghan; Meyer, James; Xu, Yang; Salyers, Kevin; Fielden, Mark; Véniant, Murielle M; Gu, Wei

    2013-12-27

    Acetyl-CoA carboxylase (ACC) is a target of interest for the treatment of metabolic syndrome. Starting from a biphenyloxadiazole screening hit, a series of piperazine oxadiazole ACC inhibitors was developed. Initial pharmacokinetic liabilities of the piperazine oxadiazoles were overcome by blocking predicted sites of metabolism, resulting in compounds with suitable properties for further in vivo studies. Compound 26 was shown to inhibit malonyl-CoA production in an in vivo pharmacodynamic assay and was advanced to a long-term efficacy study. Prolonged dosing with compound 26 resulted in impaired glucose tolerance in diet-induced obese (DIO) C57BL6 mice, an unexpected finding.

  20. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  1. Coordinate expression of hydrogenase and ribulose bisphosphate carboxylase in Rhizobium japonicum Hupc mutants.

    PubMed Central

    Merberg, D; Maier, R J

    1984-01-01

    In contrast to the wild type, H2 uptake-constitutive mutants of Rhizobium japonicum expressed both hydrogenase and ribulose bisphosphate carboxylase activities when grown heterotrophically. However, as bacteroids from soybean root nodules, the H2 uptake-constitutive mutants, like the wild type, did not express ribulose bisphosphate carboxylase activity. PMID:6384199

  2. A distinct holoenzyme organization for two-subunit pyruvate carboxylase

    PubMed Central

    Choi, Philip H.; Jo, Jeanyoung; Lin, Yu-Cheng; Lin, Min-Han; Chou, Chi-Yuan; Dietrich, Lars E. P.; Tong, Liang

    2016-01-01

    Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the β subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4β4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2β4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis. PMID:27708276

  3. Structure of the acetophenone carboxylase core complex: prototype of a new class of ATP-dependent carboxylases/hydrolases

    PubMed Central

    Weidenweber, Sina; Schühle, Karola; Demmer, Ulrike; Warkentin, Eberhard; Ermler, Ulrich; Heider, Johann

    2017-01-01

    Degradation of the aromatic ketone acetophenone is initiated by its carboxylation to benzoylacetate catalyzed by acetophenone carboxylase (Apc) in a reaction dependent on the hydrolysis of two ATP to ADP and Pi. Apc is a large protein complex which dissociates during purification into a heterooctameric Apc(αα′βγ)2 core complex of 482 kDa and Apcε of 34 kDa. In this report, we present the X-ray structure of the Apc(αα′βγ)2 core complex from Aromatoleum aromaticum at ca. 3 Å resolution which reveals a unique modular architecture and serves as model of a new enzyme family. Apcβ contains a novel domain fold composed of two β-sheets in a barrel-like arrangement running into a bundle of eight short polyproline (type II)-like helical segments. Apcα and Apcα′ possess ATP binding modules of the ASKHA superfamily integrated into their multidomain structures and presumably operate as ATP-dependent kinases for acetophenone and bicarbonate, respectively. Mechanistic aspects of the novel carboxylation reaction requiring massive structural rearrangements are discussed and criteria for specifically annotating the family members Apc, acetone carboxylase and hydantoinase are defined. PMID:28054554

  4. Effect of Light and NO3− on Wheat Leaf Phosphoenolpyruvate Carboxylase Activity

    PubMed Central

    Le Van Quy; Foyer, Christine; Champigny, Marie-Louise

    1991-01-01

    Phosphoenolpyruvate carboxylase (PEPcase) activity was studied in excised leaves of wheat (Triticum aestivum L.) in the dark and in the light, in presence of either N-free (low-NO3− leaves) or 40 millimolar KNO3 (high-NO3− leaves) nutrient solutions. PEPcase activity increased to 2.7-fold higher than that measured in dark-adapted tissue (control) during the first 60 minutes and continued to increase more slowly to 3.8-fold that of the control. This level was reached after 200 minutes exposure of the leaves to light and high NO3−. In contrast, the lower rate of increase recorded for low-NO3− leaves ceased after 60 minutes of exposure to light at 2.3-fold the control level. The short-term NO3− effect increased linearly with the level of NO3− uptake. In immunoprecipitation experiments, the antibody concentration for PEPcase precipitation increased with the protein extracts from the different treatments in the order: control, illuminated low-NO3− leaves, illuminated high-NO3− leaves. This order also applied with regard to a decreasing sensitivity to malate and an increasing stimulation by okadaic acid (an inhibitor of P-protein phosphatases). Following these studies, 32P labeling experiments were carried out in vivo. These showed that the light-induced change in the properties of the PEPcase was due to an alteration in the phosphorylation state of the protein and that this effect was enhanced in high-NO3− conditions. Based on the responses of PEPcase and sucrose phosphate synthase in wheat leaves to light and NO3−, an interpretation of the role of NO3− as either an inhibitor of P-protein phosphatase(s) or activator of protein kinase(s) is inferred. In the presence of NO3−, the phosphorylation state of both PEPcase and sucrose phosphate synthase is increased. This causes activation of the former enzyme and inhibition of the latter. We suggest that NO3− modulates the relative protein kinase/protein phosphatase ratio to favor increased

  5. Phosphoenolpyruvate carboxylase protein kinase from developing castor oil seeds: partial purification, characterization, and reversible control by photosynthate supply.

    PubMed

    Murmu, Jhadeswar; Plaxton, William C

    2007-10-01

    Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) protein kinase (PPCK) was purified approximately 1,500-fold from developing castor oil seeds (COS). Gel filtration and immunoblotting with anti-(rice PPCK2)-immune serum indicated that this Ca2+-insensitive PPCK exists as a 31-kDa monomer. COS PPCK-mediated rephosphorylation of the 107-kDa subunit (p107) of COS PEPC1 (Km = 2.2 microM) activated PEPC1 by approximately 80% when assayed under suboptimal conditions (pH 7.3, 0.2 mM PEP, and 0.125 mM malate). COS PPCK displayed remarkable selectivity for phosphorylating COS PEPC1 (relative to tobacco, sorghum, or maize PEPCs), exhibited a broad pH-activity optima of approximately pH 8.5, and at pH 7.3 was activated 40-65% by 1 mM PEP, or 10 mM Gln or Asn, but inhibited 65% by 10 mM L-malate. The possible control of COS PPCK by disulfide-dithiol interconversion was suggested by its rapid inactivation and subsequent reactivation when incubated with oxidized glutathione and then dithiothreitol. In vitro PPCK activity correlated with in vivo p107 phosphorylation status, with both peaking in mid-cotyledon to full-cotyledon developing COS. Notably, PPCK activity and p107 phosphorylation of developing COS were eliminated following pod excision or prolonged darkness of intact plants. Both effects were fully reversed 12 h following reillumination of darkened plants. These results implicate a direct relationship between the up-regulation of COS PPCK and p107 phosphorylation during the recommencement of photosynthate delivery from illuminated leaves to the non-photosynthetic COS. Overall, the results support the hypothesis that PEPC and PPCK participate in the control of photosynthate partitioning into C-skeletons needed as precursors for key biosynthetic pathways of developing COS.

  6. Mechanism of action of ribulose bisphosphate carboxylase/oxygenase.

    PubMed

    Lane, M D; Miziorko, H M

    1978-01-01

    RuBP carboxylase-oxygenase appears to catalyze carboxylation and oxygenation by homologous mechanisms. A common binding site exists on the enzyme for the acceptor substrate, RuBP. A mechanism is proposed whereby RuBP is isomerized, and a carbanion is generated at C2. Then, either CO2 or O2 is added as an electrophile at C2 to form the corresponding 3-keto-2-carboxy-RBP or 3-keto-2-hydroperoxy-RBP adduct. Hydrolytic cleavage at the C2-C3 bonds of these intermediates by the enzyme is envisioned to produce 2 molecules of 3-phosphoglycerate in the carboxylation sequence and 1 molecule of phosphoglycolate and 1 molecule of 3-phosphoglycerate in the oxygenation sequence. Further work will be necessary to establish the validity of the proposed mechanism.

  7. Ribulose diphosphate carboxylase/oxygenase. III. Isolation and properties.

    PubMed

    Ryan, F J; Tolbert, N E

    1975-06-10

    Similarities in properties of ribulose diphosphate carboxylase and oxygenase activities further substantiate the hypothesis that the same protein catalyzes both reactions. The Km (ribulose diphosphate) is 0.33 mM for the ribulose diphosphate oxygenase, when assayed in air with an oxygen electrode. Maximum activity is obtained with 10 to 35 mM MgCl2. Higher MgCl2 concentrations are inhibitory, but they shift the pH optimum from 9.3 or 9.4 to 8.7 or 9.0. MnCl2 is an effective cofactor of the oxygenase and some activity is obtained with CoCl2. Both the ribulose diphosphate carboxylase and oxygenase activity of the purified protein from spinach leaves are slowly inactivated by storage at 0 degrees and reactivated in 10 min at 50 degrees, provided both 25 mM MgCl2 and 1 mM dithiothreitol are present. The sulfhydryl groups of the enzyme which react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) are approximately 4 at pH 7.8 and 11 at pH 9.4. At both pH values ribulose diphosphate prevents two of these sulfhydryl groups from reacting with this reagent. About 50% inhibition of the oxygenase activity at pH 9.0 occurs with 50 mM bicarbonate in the presence of 3 mM ribulose diphosphate, and from variations in these parameters the inhibition is attributed to the CO2 species. The purified enzyme of acrylamide gels prevented the reduction of nitroblue tetrazolium in the presence of the superoxide radical, but the enzyme in solution did not react as a superoxide dismutase.

  8. Mitochondrial storage form of acetyl CoA carboxylase in fasted and alloxan diabetic rats

    SciTech Connect

    Roman-Lopez, C.R.; Allred, J.B.

    1986-05-01

    Sodium dodecyl sulfate-denatured biotinyl proteins will bind (/sup 14/C)methyl avidin which remains bound through polyacrylamide gel electrophoresis. The method has been used to demonstrate the presence of two high molecular weight subunit forms of acetyl CoA carboxylase in rat liver cytoplasm, both of which are precipitated by antibody to purifed rat liver acetyl CoA carboxylase prepared from sheep serum. Rat liver mitochondria contained five distinct biotinyl protein subunits, the two largest of which have been identified as acetyl CoA carboxylase subunits on the basis of precipitation by anti-acetyl CoA carboxylase antibody. The small quantity of acetyl CoA carboxylase associated with rat liver microsomes could be attributed to cytoplasmic contamination. The binding of radioactive avidin is sufficiently tight to use as a measure of the quantity of acetyl CoA carboxylase. The quantity and activity of the cytoplasmic enzyme was reduced in fasted and in alloxan diabetic rats compared to that in fed controls but the quantity of the enzyme associated with isolated mitochondria was not reduced. The results indicate that there is a mitochondrial storage form of acetyl CoA carboxylase.

  9. Phosphoenolpyruvate carboxylase regulation in C4-PEPC-expressing transgenic rice during early responses to drought stress.

    PubMed

    Liu, Xiaolong; Li, Xia; Zhang, Chen; Dai, Chuanchao; Zhou, Jiayu; Ren, Chenggang; Zhang, Jinfei

    2017-02-01

    Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) has important functions in C4 photosynthesis and biosynthesis of intermediate metabolites. In this study, the drought resistance of C4-PEPC-expressing transgenic rice (Oryza sativa, line PC) plants was assessed using simulated drought conditions [i.e. polyethylene glycol (PEG)-6000 treatment]. The dry weight of PC plants was higher than that of wild-type (WT) plants following treatment with 15% PEG-6000 for 16 days. Furthermore, the water use efficiency, relative water content and proline content in PC plants were higher than those of WT plants, as were C4-PEPC activity and transcript levels following treatment with 5% PEG-6000 for 2 h. The protein kinase activities and transcript levels of sucrose non-fermenting-1-related protein kinases (SnRKs) genes, such as SnRK1a, OsK24 and OsK35 were also higher in PC plants than in WT plants following treatment with 5% PEG-6000 for 2 h. Additionally, phosphoenolpyruvate carboxylase kinase (PPCK, EC 4.1.1.32) activities and transcript levels (e.g. PPCK1 and PPCK2) increased following drought treatment. These changes were regulated by signaling molecules, such as calcium, nitric oxide and hydrogen peroxide. Furthermore, the -1095 to -416 region of the C4-PEPC promoter in PC plants was demethylated following exposure to drought conditions for 1 h. The demethylation coincided with an increase in C4-PEPC expression. Our data suggest that the demethylation of the C4-PEPC promoter and the phosphorylation catalyzed by PPCK have key roles in conferring drought tolerance to the transgenic rice plants.

  10. In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds

    PubMed Central

    Echevarría, Cristina

    2014-01-01

    Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an important cytosolic regulatory enzyme that plays a pivotal role in numerous physiological processes in plants, including seed development and germination. Previous studies demonstrated the occurrence of immunoreactive PEPC polypeptides of ~110kDa and 107kDa (p110 and p107, respectively) on immunoblots of clarified extracts of germinating sorghum (Sorghum bicolor) seeds. In order to establish the biochemical basis for this observation, a 460kDa PEPC heterotetramer composed of an equivalent ratio of p110 and p107 subunits was purified to near homogeneity from the germinated seeds. Mass spectrometry established that p110 and p107 are both encoded by the same plant-type PEPC gene (CP21), but that p107 was in vivo monoubiquitinated at Lys624 to form p110. This residue is absolutely conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Anti-ubiquitin IgG immunodetected p110 but not p107, whereas incubation with a deubiquitinating enzyme (USP-2 core) efficiently converted p110 into p107, while relieving the enzyme’s feedback inhibition by l-malate. Partial PEPC monoubiquitination was also detected during sorghum seed development. It is apparent that monoubiquitination at Lys624 is opposed to phosphorylation at Ser7 in terms of regulating the catalytic activity of sorghum seed PEPC. PEPC monoubiquitination is hypothesized to fine-tune anaplerotic carbon flux according to the cell’s immediate physiological requirements for tricarboxylic acid cycle intermediates needed in support of biosynthesis and carbon–nitrogen interactions. PMID:24288181

  11. Acetyl CoA carboxylase inactivation and meiotic maturation in mouse oocytes.

    PubMed

    Valsangkar, Deepa S; Downs, Stephen M

    2015-09-01

    In mouse oocytes, meiotic induction by pharmacological activation of PRKA (adenosine monophosphate-activated protein kinase; formerly known as AMPK) or by hormones depends on stimulation of fatty acid oxidation (FAO). PRKA stimulates FAO by phosphorylating and inactivating acetyl CoA carboxylase (ACAC; formerly ACC), leading to decreased malonyl CoA levels and augmenting fatty-acid transport into mitochondria. We investigated a role for ACAC inactivation in meiotic resumption by testing the effect of two ACAC inhibitors, CP-640186 and Soraphen A, on mouse oocytes maintained in meiotic arrest in vitro. These inhibitors significantly stimulated the resumption of meiosis in arrested cumulus cell-enclosed oocytes, denuded oocytes, and follicle-enclosed oocytes. This stimulation was accompanied by an increase in FAO. Etomoxir, a malonyl CoA analogue, prevented meiotic resumption as well as the increase in FAO induced by ACAC inhibition. Citrate, an ACAC activator, and CBM-301106, an inhibitor of malonyl CoA decarboxylase, which converts malonyl CoA to acetyl CoA, suppressed both meiotic induction and FAO induced by follicle-stimulating hormone, presumably by maintaining elevated malonyl CoA levels. Mouse oocyte-cumulus cell complexes contain both isoforms of ACAC (ACACA and ACACB); when wild-type and Acacb(-/-) oocytes characteristics were compared, we found that these single-knockout oocytes showed a significantly higher FAO level and a reduced ability to maintain meiotic arrest, resulting in higher rates of germinal vesicle breakdown. Collectively, these data support the model that ACAC inactivation contributes to the maturation-promoting activity of PRKA through stimulation of FAO.

  12. Coordinating Role of His216 in MgATP Binding and Cleavage in Pyruvate Carboxylase

    PubMed Central

    2015-01-01

    His216 is a well-conserved residue in pyruvate carboxylases and, on the basis of structures of the enzyme, appears to have a role in the binding of MgATP, forming an interaction with the 3′-hydroxyl group of the ribose ring. Mutation of this residue to asparagine results in a 9-fold increase in the Km for MgATP in its steady-state cleavage in the absence of pyruvate and a 3-fold increase in the Km for MgADP in its steady-state phosphorylation by carbamoyl phosphate. However, from single-turnover experiments of MgATP cleavage, the Kd of the enzyme·MgATP complex is essentially the same in the wild-type enzyme and H216N. Direct stopped-flow measurements of nucleotide binding and release using the fluorescent analogue FTP support these observations. However, the first-order rate constant for MgATP cleavage in the single-turnover experiments in H216N is only 0.75% of that for the wild-type enzyme, and thus, the MgATP cleavage step is rate-limiting in the steady state for H216N but not for the wild-type enzyme. Close examination of the structure of the enzyme suggested that His216 may also interact with Glu218, which in turn interacts with Glu305 to form a proton relay system involved in the deprotonation of bicarbonate. Single-turnover MgATP cleavage experiments with mutations of these two residues resulted in kinetic parameters similar to those observed in H216N. We suggest that the primary role of His216 is to coordinate the binding of MgATP and the deprotonation of bicarbonate in the reaction to form the putative carboxyphosphate intermediate by participation in a proton relay system involving Glu218 and Glu305. PMID:24460480

  13. Chemical and Physical Characterization of the Activation of Ribulosebiphosphate Carboxylase/Oxygenase

    DOE R&D Accomplishments Database

    Donnelly, M. I.; Ramakrishnan, V.; Hartman, F. C.

    1983-08-01

    Molecular structure of ribulosebiphosphate carboxylase/oxygenase isolated from Rhodospirillium was compared with the enzyme isolated from Alcaligens eutrophus. Peptides derived from the active center of the bacterial enzyme were highly homologous with those isolated from spinach. Molecular shapes of the carboxylases were estimated using neutron scattering data. These studies suggested that the enzyme as isolated from R. rubrum is a solid prolate ellipsoid or cylinder, while the spinach enzyme resembles a hollow sphere.

  14. Crystallization and structure of a recombinant ribulose-1,5-bisphosphate carboxylase

    NASA Astrophysics Data System (ADS)

    Schneider, Gunter; Lindqvist, Ylva; Brändén, Carl-Ivar; Lorimer, George

    1988-07-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase is the key enzyme in photosynthetic carbon dioxide fixation and photorespiration. The dimeric carboxylase from the photosynthetic bacterium Rhodospirillum rubrum has been cloned and expressed in E. coli. The recombinant enzyme has been crystallized in a number of different crystal forms. The three-dimensional structure of the enzyme has been determined by X-ray crystallographic methods to 2.9Åresolution.

  15. Variations in Km(CO2) of Ribulose-1,5-bisphosphate Carboxylase among Grasses

    PubMed Central

    Yeoh, Hock-Hin; Badger, Murray R.; Watson, Leslie

    1980-01-01

    A survey of the Km(CO2) values of ribulose-1,5-bisphosphate carboxylase from 60 grass species shows that enzyme from C3 grasses consistently exhibits lower Km(CO2) than does that from C4 grasses. Systematically ordered variation in Km(CO2) of ribulose-1,5-bisphosphate carboxylases from C3 and C4 grasses is also apparent and, among C4 grasses, this shows some correlation with C4 types. PMID:16661586

  16. Chemical and physical characterization of the activation of ribulosebiphosphate carboxylase/oxygenase

    SciTech Connect

    Donnelly, M.I.; Ramakrishnan, V.; Hartman, F.C.

    1983-01-01

    Molecular structure of ribulosebiphosphate carboxylase/oxygenase isolated from Rhodospirillium was compared with the enzyme isolated from Alcaligens eutrophus. Peptides derived from the active center of the bacterial enzyme were highly homologous with those isolated from spinach. Molecular shapes of the carboxylases were estimated using neutron scattering data. These studies suggested that the enzyme as isolated from R. rubrum is a solid prolate ellipsoid or cylinder, while the spinach enzyme resembles a hollow sphere. 1 drawing.

  17. Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase.

    PubMed

    Broussard, Tyler C; Pakhomova, Svetlana; Neau, David B; Bonnot, Ross; Waldrop, Grover L

    2015-06-23

    Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1'-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1'-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO₂ from the carboxyphosphate intermediate to biotin.

  18. Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum

    PubMed Central

    Koffas, Mattheos A. G.; Jung, Gyoo Yeol; Aon, Juan C.; Stephanopoulos, Gregory

    2002-01-01

    Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology. PMID:12406733

  19. Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

  20. [Phosphorylation of tau protein].

    PubMed

    Uchida, T; Ishiguro, K

    1990-05-01

    In aged human brain and particularly in Alzheimer's disease brain, paired helical filaments (PHFs) accumulate in the neuronal cell. Recently, it has been found that the highly phosphorylated tau protein, one of the microtubule-associated proteins (MAPs), is a component of PHF. The authors attempted to clarify the mechanism underlying the accumulation of PHF from the following two aspects; 1) What is the mechanism of phosphorylation of tau protein? 2) Is the highly phosphorylated tau protein capable of forming PHFs? From rat or bovine microtubule proteins we partially purified and characterized a novel protein kinase that specifically phosphorylated tau and MAP2 among many proteins in the brain extract, and which formed a PHF epitope on the phosphorylated human tau. This enzyme was one of the protein serine/threonine kinases and was independent of known second messengers. The phosphorylation of tau by this enzyme was stimulated by tubulin under the condition of microtubule formation, suggesting that the phosphorylation of tau could occur concomitantly with microtubule formation in the brain. Since this kinase was usually bound to tau but not directly to tubulin, the enzyme was associated with microtubules through tau. From these properties related to tau, this kinase is designated as tau protein kinase. The tau that been phosphorylated with this kinase using [gamma-32P]ATP as a phosphate donor, was digested by endoprotinase Lys-C to produce three labeled fragments, K1, K2 and K3. These three fragments were sequenced and the phosphorylation sites on tau by this kinase were identified. The K2 fragment overlapped with the tau-1 site known to be one of the phosphorylation site in PHF. This result strengthens the possibility that tau protein phosphorylated by tau protein kinase is incorporated into PHF. Tubulin binding sites on tau were located between K1 and K3 fragments, while K2 fragment was located in the neighboring to N-terminus of K1. No phosphorylated sites were

  1. Cloning, expression, purification and physical and kinetic characterization of the phosphoenolpyruvate carboxylase from orange (Citrus sinensis osbeck var. Valencia) fruit juice sacs.

    PubMed

    Perotti, Valeria E; Figueroa, Carlos M; Andreo, Carlos S; Iglesias, Alberto A; Podestá, Florencio E

    2010-11-01

    Phosphoenolpyruvate (PEP) carboxylase (PEPCase) from orange fruit juice sacs has been cloned and heterogously expressed in high yield. The purified recombinant enzyme displays properties typical of plant PEPCase, including activation by sugar phosphates and inhibition by malate and citrate. Malate inhibition is weak in the physiological pH range, and the enzyme is also poorly affected by Glu and Asp, known inhibitors of C(3) plants PEPCases. However, it is strongly inhibited by citrate. Orange fruit PEPCase phosphorylation by mammalian protein kinase A decreased inhibition by malate. The enzyme presents an unusual high molecular mass in the absence of PEP, while in its presence it displays a more common tetrameric arrangement. The overall properties of the enzyme suggest that it is suited for organic acid synthesis and NADH reoxidation in the mature fruit. The present study provides the first analysis of a recombinant fruit PEPCase.

  2. Resistance to acetyl-CoA carboxylase-inhibiting herbicides.

    PubMed

    Kaundun, Shiv S

    2014-09-01

    Resistance to acetyl-CoA carboxylase herbicides is documented in at least 43 grass weeds and is particularly problematic in Lolium, Alopecurus and Avena species. Genetic studies have shown that resistance generally evolves independently and can be conferred by target-site mutations at ACCase codon positions 1781, 1999, 2027, 2041, 2078, 2088 and 2096. The level of resistance depends on the herbicides, recommended field rates, weed species, plant growth stages, specific amino acid changes and the number of gene copies and mutant ACCase alleles. Non-target-site resistance, or in essence metabolic resistance, is prevalent, multigenic and favoured under low-dose selection. Metabolic resistance can be specific but also broad, affecting other modes of action. Some target-site and metabolic-resistant biotypes are characterised by a fitness penalty. However, the significance for resistance regression in the absence of ACCase herbicides is yet to be determined over a practical timeframe. More recently, a fitness benefit has been reported in some populations containing the I1781L mutation in terms of vegetative and reproductive outputs and delayed germination. Several DNA-based methods have been developed to detect known ACCase resistance mutations, unlike metabolic resistance, as the genes remain elusive to date. Therefore, confirmation of resistance is still carried out via whole-plant herbicide bioassays. A growing number of monocotyledonous crops have been engineered to resist ACCase herbicides, thus increasing the options for grass weed control. While the science of ACCase herbicide resistance has progressed significantly over the past 10 years, several avenues provided in the present review remain to be explored for a better understanding of resistance to this important mode of action.

  3. Light Modulation of Maize Leaf Phosphoenolpyruvate Carboxylase 1

    PubMed Central

    Huber, Steven C.; Sugiyama, Tatsuo; Akazawa, Takashi

    1986-01-01

    Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH4)2SO4 fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition. Images Fig. 6 PMID:16665065

  4. Pyruvate Carboxylase Activates the RIG-I-like Receptor-Mediated Antiviral Immune Response by Targeting the MAVS signalosome

    PubMed Central

    Cao, Zhongying; Zhou, Yaqin; Zhu, Shengli; Feng, Jian; Chen, Xueyuan; Liu, Shi; Peng, Nanfang; Yang, Xiaodan; Xu, Gang; Zhu, Ying

    2016-01-01

    When retinoic acid-inducible gene 1 protein (RIG-I)-like receptors sense viral dsRNA in the cytosol, RIG-I and melanoma differentiation-associated gene 5 (MDA5) are recruited to the mitochondria to interact with mitochondrial antiviral signaling protein (MAVS) and initiate antiviral immune responses. In this study, we demonstrate that the biotin-containing enzyme pyruvate carboxylase (PC) plays an essential role in the virus-triggered activation of nuclear factor kappa B (NF-κB) signaling mediated by MAVS. PC contributes to the enhanced production of type I interferons (IFNs) and pro-inflammatory cytokines, and PC knockdown inhibits the virus-triggered innate immune response. In addition, PC shows extensive antiviral activity against RNA viruses, including influenza A virus (IAV), human enterovirus 71 (EV71), and vesicular stomatitis virus (VSV). Furthermore, PC mediates antiviral action by targeting the MAVS signalosome and induces IFNs and pro-inflammatory cytokines by promoting phosphorylation of NF-κB inhibitor-α (IκBα) and the IκB kinase (IKK) complex, as well as NF-κB nuclear translocation, which leads to activation of interferon-stimulated genes (ISGs), including double-stranded RNA-dependent protein kinase (PKR) and myxovirus resistance protein 1 (Mx1). Our findings suggest that PC is an important player in host antiviral signaling. PMID:26906558

  5. Crystallization and characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from eight plant species.

    PubMed

    Johal, S; Bourque, D P; Smith, W W; Suh, S W; Eisenberg, D

    1980-09-25

    Ribulose bisphosphate carboxylase/oxygenase was isolated and crystallized from eight plant species. Crystals grew from either of two similar sets of crystallizing conditions: crystals of the enzyme from alfalfa, corn, cotton, potato, spinach, tobacco, and tomato grew from solutions containing phosphate and polyethylene glycol 6000 as a precipitant, and those from potato, tobacco (both Nicotiana sylvestris and Nicotiana tabacum), and tomato grew from a mixture of ammonium sulfate and phosphate. Crystals of the enzyme from potato and both species of tobacco were large enough to characterize by x-ray diffraction and were found to have the Form III structure, previously reported for crystals of ribulose bisphosphate carboxylase/oxygenase from N. tabacum. For crystalline material from several species, both carboxylase and oxygenase activites have been assayed and copper and iron contents have been determined. The possible significance of the observed general conditions of crystallization of this enzyme is discussed.

  6. Changing ribulose diphosphate carboxylase/oxygenase activity in ripening tomato fruit.

    PubMed

    Bravdo, B A; Palgi, A; Lurie, S

    1977-08-01

    Tomato fruit (Lycopersicum esculentum Mill) from green, pink, and red stages were assayed for changes in the activity of ribulose diphosphate carboxylase and oxygenase, phosphoenolpyruvate carboxylase, changes in the levels of glycolate and respiratory gas exchange. The ribulose diphosphate carboxylase activity decreased as the fruit ripened. By comparison, the ribulose diphosphate oxygenase activity increased during the transition from the green to the pink stage, and declined afterward. The changes in the endogenous glycolate levels and the respiratory gas exchange, as observed at different stages of ripening, resembled the changes in the ribulose diphosphate oxygenase activity. The utilization of glycolate in further metabolic activity may result in the formation of peroxidases required for the onset of ripening.

  7. NP-40 reduces contamination by endogenous biotinylated carboxylases during purification of biotin tagged nuclear proteins.

    PubMed

    Papageorgiou, Dimitris N; Demmers, Jeroen; Strouboulis, John

    2013-05-01

    We describe here a simple procedure for greatly reducing contamination of nuclear extracts by naturally biotinylated cytoplasmic carboxylases, which represent a major source of non-specific background when employing BirA-mediated biotinylation tagging for the purification and characterization of nuclear protein complexes by mass spectrometry. We show that the use of 0.5% of the non-ionic detergent Nonidet-40 (NP-40) during cell lysis and nuclei isolation is sufficient to practically eliminate contamination of nuclear extracts by carboxylases and to greatly reduce background signals in downstream mass spectrometric analyses.

  8. Phosphorylation: Implications in Cancer.

    PubMed

    Singh, Vishakha; Ram, Mahendra; Kumar, Rajesh; Prasad, Raju; Roy, Birendra Kumar; Singh, Kaushal Kumar

    2017-02-01

    Post translational modifications (PTMs) are involved in variety of cellular activities and phosphorylation is one of the most extensively studied PTM, which regulates a number of cellular functions like cell growth, differentiation, apoptosis and cell signaling in healthy condition. However, alterations in phosphorylation pathways result in serious outcomes in the form of diseases, especially cancer. Many signalling pathways including Tyrosine kinase, MAP kinase, Cadherin-catenin complex, Cyclin-dependent kinase etc. are major players of the cell cycle and deregulation in their phosphorylation-dephosphorylation cascade has been shown to be manifested in the form of various types of cancers. Tyrosine kinase family encompasses the greatest number of oncoproteins. MAPK cascade has an importance role in cancer growth and progression. Bcl-2 family proteins serve either proapoptotic or antiapoptotic function. Cadherin-catenin complex regulates cell adhesion properties and cyclins are the key regulators of cell cycle. Altered phosphorylations in any of the above pathways are strongly associated with cancer, at the same time they serve as the potential tergets for drug development against cancer. Drugs targeting tyrosine kinase are potent anticancer drugs. Inhibitors of MEK, PI3K and ERK signalling pathways are undergoing clinical trials. Thus, drugs targeting phosphorylation pathways represent a promising area for cancer therapy.

  9. Identification and expression of a soybean nodule-enhanced PEP-carboxylase kinase gene (NE-PpcK) that shows striking up-/down-regulation in vivo.

    PubMed

    Xu, Wenxin; Zhou, You; Chollet, Raymond

    2003-05-01

    Various isoforms of plant phosphoenolpyruvate carboxylase (PEPC (Ppc)) are controlled post-translationally by an intricate interaction between allosteric regulation and reversible protein phosphorylation. In leaves and root nodules of legumes, these changes in PEPC phosphorylation state are governed primarily by PEPC-kinase (PpcK), a novel, 'minimal but functional' Ser/Thr kinase. To date, this plant-specific kinase has been investigated in molecular terms exclusively in non-leguminous plants, such as Crassulacean-acid-metabolism (CAM) species and Arabidopsis. As an important extension of our earlier biochemical studies on this dedicated kinase and PEPC phosphorylation in soybean (Glycine max) nodules, we now report the molecular cloning of the first legume PpcK from a soybean nodule cDNA library, which encodes a functional, 31.0 kDa PpcK polypeptide. Besides displaying organ, developmental, and spatial expression properties that are strikingly up-regulated in mature nodules, the expression pattern of this transcript is distinct from that of a second soybean PpcK isogene (GmPpcK). The steady-state abundance of this former, nodule-enhanced transcript (NE-PpcK) is markedly influenced by photosynthate supply from the shoots. This latter up-/down-regulation of NE-PpcK transcript level occurs in vivo in concert with the corresponding changes in the nodule PpcK activity, the phosphorylation-state of PEPC, and the abundance of a previously identified, nodule-enhanced transcript (GmPEPC7) that encodes the target enzyme (NE-Ppc). Furthermore, genomic Southern analysis and inspection of the public database indicate that there are at least three distinct PpcK and Ppc isogenes in soybean. Collectively, these and recent findings with Arabidopsis implicate the existence of multiple PpcK-Ppc'expression-partners' in plants, exemplified by NE-PpcK and NE-Ppc in the soybean nodule.

  10. Fenofibrate activates AMPK and increases eNOS phosphorylation in HUVEC

    SciTech Connect

    Murakami, Hisashi; Murakami, Ryuichiro . E-mail: ryuichi@med.nagoya-u.ac.jp; Kambe, Fukushi; Cao, Xia; Takahashi, Ryotaro; Asai, Toru; Hirai, Toshihisa; Numaguchi, Yasushi; Okumura, Kenji; Seo, Hisao; Murohara, Toyoaki

    2006-03-24

    Fenofibrate improves endothelial function by lipid-lowering and anti-inflammatory effects. Additionally, fenofibrate has been demonstrated to upregulate endothelial nitric oxide synthase (eNOS). AMP-activated protein kinase (AMPK) has been reported to phosphorylate eNOS at Ser-1177 and stimulate vascular endothelium-derived nitric oxide (NO) production. We report here that fenofibrate activates AMPK and increases eNOS phosphorylation and NO production in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with fenofibrate increased the phosphorylation of AMPK and acetyl-CoA carboxylase. Fenofibrate simultaneously increased eNOS phosphorylation and NO production. Inhibitors of protein kinase A and phosphatidylinositol 3-kinase failed to suppress the fenofibrate-induced eNOS phosphorylation. Neither bezafibrate nor WY-14643 activated AMPK in HUVEC. Furthermore, fenofibrate activated AMPK without requiring any transcriptional activities. These results indicate that fenofibrate stimulates eNOS phosphorylation and NO production through AMPK activation, which is suggested to be a novel characteristic of this agonist and unrelated to its effects on peroxisome proliferator-activated receptor {alpha}.

  11. Isolation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a multi-functional enzyme that catalyzes the fixation of CO2 and O2 in photosynthesis and photorespiration, respectively. As the rate-limiting step in photosynthesis, improving the catalytic properties of Rubisco has long been viewed as a...

  12. Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes.

    PubMed

    Gamberino, W C; Berkich, D A; Lynch, C J; Xu, B; LaNoue, K F

    1997-12-01

    CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C-labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C] glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate --> alpha-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or

  13. Crystal Structure of the alpha6beta6 Holoenzyme of propionyl-coenzyme A Carboxylase

    SciTech Connect

    Huang, C.; Sadre-Bazzaz, K; Shen, Y; Deng, B; Zhou, Z; Tong, L

    2010-01-01

    Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an {alpha}{sub 6}{beta}{sub 6} dodecamer, with a molecular mass of 750 kDa. The {alpha}-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the {beta}-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-{angstrom} resolution of a bacterial PCC {alpha}{sub 6}{beta}{sub 6} holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-{angstrom} resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the {alpha}-subunits are arranged as monomers in the holoenzyme, decorating a central {beta}{sub 6} hexamer. A hitherto unrecognized domain in the {alpha}-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the {beta}-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 {angstrom}, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the {beta}-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).

  14. Acute renal ischemia rapidly activates the energy sensor AMPK but does not increase phosphorylation of eNOS-Ser1177.

    PubMed

    Mount, Peter F; Hill, Rebecca E; Fraser, Scott A; Levidiotis, Vicki; Katsis, Frosa; Kemp, Bruce E; Power, David A

    2005-11-01

    A fundamental aspect of acute renal ischemia is energy depletion, manifest as a falling level of ATP that is associated with a simultaneous rise in AMP. The energy sensor AMP-activated protein kinase (AMPK) is activated by a rising AMP-to-ATP ratio, but its role in acute renal ischemia is unknown. AMPK is activated in the ischemic heart and is reported to phosphorylate both endothelial nitric oxide synthase (eNOS) and acetyl-CoA carboxylase. To study activation of AMPK in acute renal ischemia, the renal pedicle of anesthetized Sprague-Dawley rats was cross-clamped for increasing time intervals. AMPK was strongly activated within 1 min and remained so after 30 min. However, despite the robust activation of AMPK, acute renal ischemia did not increase phosphorylation of the AMPK phosphorylation sites eNOS-Ser(1177) or acetyl-CoA carboxylase-Ser(79). Activation of AMPK in bovine aortic endothelial cells by the ATP-depleting agent antimycin A and the antidiabetic drug phenformin also did not increase phosphorylation of eNOS-Ser(1177), confirming that AMPK activation and phosphorylation of eNOS are dissociated in some situations. Immunoprecipitation studies demonstrated that the dissociation between AMPK activation and phosphorylation of eNOS-Ser(1177) was not due to changes in the physical associations between AMPK, eNOS, or heat shock protein 90. In conclusion, acute renal ischemia rapidly activates the energy sensor AMPK, which is known to maintain ATP reserves during energy stress. The substrates it phosphorylates, however, are different from those in other organs such as the heart.

  15. Phosphorylation regulates mycobacterial proteasome.

    PubMed

    Anandan, Tripti; Han, Jaeil; Baun, Heather; Nyayapathy, Seeta; Brown, Jacob T; Dial, Rebekah L; Moltalvo, Juan A; Kim, Min-Seon; Yang, Seung Hwan; Ronning, Donald R; Husson, Robert N; Suh, Joowon; Kang, Choong-Min

    2014-09-01

    Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.

  16. Struvite and prebiotic phosphorylation.

    NASA Technical Reports Server (NTRS)

    Handschuh, G. J.; Orgel, L. E.

    1973-01-01

    Struvite rather than apatite or amorphous calcium phosphate is precipitated when phosphate is added to seawater containing more than 0.01M NH4+ ions. Struvite may have precipitated from evaporating seawater on the primitive earth, and may have been important for prebiotic phosphorylation.

  17. Methylcrotonoyl-CoA carboxylase 1 potentiates RLR-induced NF-κB signaling by targeting MAVS complex

    PubMed Central

    Cao, Zhongying; Xia, Zhangchuan; Zhou, Yaqin; Yang, Xiaodan; Hao, Hua; Peng, Nanfang; Liu, Shi; Zhu, Ying

    2016-01-01

    RNA virus infections are detected by the RIG-I family of receptors, which signal through the adaptor molecule mitochondrial antiviral signaling (MAVS). MAVS then recruits the adaptor’s tumor necrosis factor receptor-associated factor (TRAF) 3 and TRAF6, which in turn activate IRF3 and NF-κB, respectively, to induce interferons (IFNs) and inflammatory responses. Here we show that the biotin-containing enzyme methylcrotonoyl-CoA carboxylase 1 (MCCC1) enhances virus-induced, MAVS-mediated IFN and inflammatory cytokine expression through the NF-κB signaling pathway. MCCC1 knockdown strongly inhibits induction of IFNs and inflammatory cytokines. Furthermore, MCCC1 shows extensive antiviral activity toward RNA viruses, including influenza A virus, human enterovirus 71, and vesicular stomatitis virus. Here, we have elucidated the mechanism underlying MCCC1-mediated inhibition of viral replication. MCCC1 interacts with MAVS and components of the MAVS signalosome and contributes to enhanced production of type I IFNs and pro-inflammatory cytokines by promoting phosphorylation of the IκB kinase (IKK) complex and NF-κB inhibitor-α (IκBα), as well as NF-κB nuclear translocation. This process leads to activation of IFNs and cytokine expression and subsequent activation of IFN-stimulated genes, including double-stranded RNA-dependent protein kinase PKR and myxovirus resistance protein 1. These findings demonstrate that MCCC1 plays an essential role in virus-triggered, MAVS-mediated activation of NF-κB signaling. PMID:27629939

  18. Computational redesign of bacterial biotin carboxylase inhibitors using structure-based virtual screening of combinatorial libraries.

    PubMed

    Brylinski, Michal; Waldrop, Grover L

    2014-04-02

    As the spread of antibiotic resistant bacteria steadily increases, there is an urgent need for new antibacterial agents. Because fatty acid synthesis is only used for membrane biogenesis in bacteria, the enzymes in this pathway are attractive targets for antibacterial agent development. Acetyl-CoA carboxylase catalyzes the committed and regulated step in fatty acid synthesis. In bacteria, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. Fragment-based screening revealed that amino-oxazole inhibits biotin carboxylase activity and also exhibits antibacterial activity against Gram-negative organisms. In this report, we redesigned previously identified lead inhibitors to expand the spectrum of bacteria sensitive to the amino-oxazole derivatives by including Gram-positive species. Using 9,411 small organic building blocks, we constructed a diverse combinatorial library of 1.2×10⁸ amino-oxazole derivatives. A subset of 9×10⁶ of these compounds were subjected to structure-based virtual screening against seven biotin carboxylase isoforms using similarity-based docking by eSimDock. Potentially broad-spectrum antibiotic candidates were selected based on the consensus ranking by several scoring functions including non-linear statistical models implemented in eSimDock and traditional molecular mechanics force fields. The analysis of binding poses of the top-ranked compounds docked to biotin carboxylase isoforms suggests that: (1) binding of the amino-oxazole anchor is stabilized by a network of hydrogen bonds to residues 201, 202 and 204; (2) halogenated aromatic moieties attached to the amino-oxazole scaffold enhance interactions with a hydrophobic pocket formed by residues 157, 169, 171 and 203; and (3) larger substituents reach deeper into the binding pocket to form additional hydrogen bonds with the side chains of residues 209 and 233. These structural insights into drug

  19. The impact of PEPC phosphorylation on growth and development of Arabidopsis thaliana: molecular and physiological characterization of PEPC kinase mutants.

    PubMed

    Meimoun, Patrice; Gousset-Dupont, Aurélie; Lebouteiller, Bénédicte; Ambard-Bretteville, Françoise; Besin, Evelyne; Lelarge, Caroline; Mauve, Caroline; Hodges, Michael; Vidal, Jean

    2009-05-19

    Two phosphoenolpyruvate carboxylase (PEPC) kinase genes (PPCk1 and PPCk2) are present in the Arabidopsis genome; only PPCk1 is expressed in rosette leaves. Homozygous lines of two independent PPCk1 T-DNA-insertional mutants showed very little (dln1), or no (csi8) light-induced PEPC phosphorylation and a clear retard in growth under our greenhouse conditions. A mass-spectrometry-based analysis revealed significant changes in metabolite profiles. However, the anaplerotic pathway initiated by PEPC was only moderately altered. These data establish the PPCk1 gene product as responsible for leaf PEPC phosphorylation in planta and show that the absence of PEPC phosphorylation has pleiotropic consequences on plant metabolism.

  20. Tissue-specific expression and post-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant, Ricinus communis L.

    PubMed

    O'Leary, Brendan; Fedosejevs, Eric T; Hill, Allyson T; Bettridge, James; Park, Joonho; Rao, Srinath K; Leach, Craig A; Plaxton, William C

    2011-11-01

    This study employs transcript profiling together with immunoblotting and co-immunopurification to assess the tissue-specific expression, protein:protein interactions, and post-translational modifications (PTMs) of plant- and bacterial-type phosphoenolpyruvate carboxylase (PEPC) isozymes (PTPC and BTPC, respectively) in the castor plant, Ricinus communis. Previous studies established that the Class-1 PEPC (PTPC homotetramer) of castor oil seeds (COS) is activated by phosphorylation at Ser-11 and inhibited by monoubiquitination at Lys-628 during endosperm development and germination, respectively. Elimination of photosynthate supply to developing COS by depodding caused the PTPC of the endosperm and cotyledon to be dephosphorylated, and then subsequently monoubiquitinated in vivo. PTPC monoubiquitination rather than phosphorylation is widespread throughout the castor plant and appears to be the predominant PTM of Class-1 PEPC that occurs in planta. The distinctive developmental patterns of PTPC phosphorylation versus monoubiquitination indicates that these two PTMs are mutually exclusive. By contrast, the BTPC: (i) is abundant in the inner integument, cotyledon, and endosperm of developing COS, but occurs at low levels in roots and cotyledons of germinated COS, (ii) shows a unique developmental pattern in leaves such that it is present in leaf buds and young expanding leaves, but undetectable in fully expanded leaves, and (iii) tightly interacts with co-expressed PTPC to form the novel and allosterically-desensitized Class-2 PEPC heteromeric complex. BTPC and thus Class-2 PEPC up-regulation appears to be a distinctive feature of rapidly growing and/or biosynthetically active tissues that require a large anaplerotic flux from phosphoenolpyruvate to replenish tricarboxylic acid cycle C-skeletons being withdrawn for anabolism.

  1. Ribulose-1,5-bisphosphate carboxylase/oxygenase from thermophilic cyanobacterium Thermosynechococcus elongatus.

    PubMed

    Gubernator, Beata; Bartoszewski, Rafal; Kroliczewski, Jaroslaw; Wildner, Guenter; Szczepaniak, Andrzej

    2008-01-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) can be divided into two branches: the "red-like type" of marine algae and the "green-like type" of cyanobacteria, green algae, and higher plants. We found that the "green-like type" rubisco from the thermophilic cyanobacterium Thermosynechococcus elongatus has an almost 2-fold higher specificity factor compared with rubiscos of mesophilic cyanobacteria, reaching the values of higher plants, and simultaneously revealing an improvement in enzyme thermostability. The difference in the activation energies at the transition stages between the oxygenase and carboxylase reactions for Thermosynechococcus elongatus rubisco is very close to that of Galdieria partita and significantly higher than that of spinach. This is the first characterization of a "green-like type" rubisco from thermophilic organism.

  2. Nonstructural 5A Protein of Hepatitis C Virus Interacts with Pyruvate Carboxylase and Modulates Viral Propagation

    PubMed Central

    Kim, Jong-Wook; Hwang, Soon B.

    2013-01-01

    Hepatitis C virus (HCV) is highly dependent on cellular factors for its own propagation. By employing tandem affinity purification method, we identified pyruvate carboxylase (PC) as a cellular partner for NS5A protein. NS5A interacted with PC through the N-terminal region of NS5A and the biotin carboxylase domain of PC. PC expression was decreased in cells expressing NS5A and HCV-infected cells. Promoter activity of PC was also decreased by NS5A protein. However, FAS expression was increased in cells expressing NS5A and cell culture grown HCV (HCVcc)-infected cells. Silencing of PC promoted fatty acid synthase (FAS) expression level. These data suggest HCV may modulate PC via NS5A protein for its own propagation. PMID:23861867

  3. Crystal Structures of Human and Staphylococcus aureus Pyruvate Carboxylase and Molecular Insights into the Carboxyltransfer Reaction

    SciTech Connect

    Xiang,S.; Tong, L.

    2008-01-01

    Pyruvate carboxylase (PC) catalyzes the biotin-dependent production of oxaloacetate and has important roles in gluconeogenesis, lipogenesis, insulin secretion and other cellular processes. PC contains the biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) domains. We report here the crystal structures at 2.8-Angstroms resolution of full-length PC from Staphylococcus aureus and the C-terminal region (missing only the BC domain) of human PC. A conserved tetrameric association is observed for both enzymes, and our structural and mutagenesis studies reveal a previously uncharacterized domain, the PC tetramerization (PT) domain, which is important for oligomerization. A BCCP domain is located in the active site of the CT domain, providing the first molecular insights into how biotin participates in the carboxyltransfer reaction. There are dramatic differences in domain positions in the monomer and the organization of the tetramer between these enzymes and the PC from Rhizobium etli.

  4. Is Dimerization Required for the Catalytic Activity of Bacterial Biotin Carboxylase?

    SciTech Connect

    Shen,Y.; Chou, C.; Chang, G.; Tong, L.

    2006-01-01

    Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism. The biotin carboxylase (BC) subunit of Escherichia coli ACC is believed to be active only as a dimer, although the crystal structure shows that the active site of each monomer is 25 Angstroms from the dimer interface. We report here biochemical, biophysical, and structural characterizations of BC carrying single-site mutations in the dimer interface. Our studies demonstrate that two of the mutants, R19E and E23R, are monomeric in solution but have only a 3-fold loss in catalytic activity. The crystal structures of the E23R and F363A mutants show that they can still form the correct dimer at high concentrations. Our data suggest that dimerization is not an absolute requirement for the catalytic activity of the E. coli BC subunit, and we propose a new model for the molecular mechanism of action for BC in multisubunit and multidomain ACCs.

  5. Phosphorylation of the transit sequence of chloroplast precursor proteins.

    PubMed

    Waegemann, K; Soll, J

    1996-03-15

    A protein kinase was located in the cytosol of pea mesophyll cells. The protein kinase phosphorylates, in an ATP-dependent manner, chloroplast-destined precursor proteins but not precursor proteins, which are located to plant mitochondria or plant peroxisomes. The phosphorylation occurs on either serine or threonine residues, depending on the precursor protein used. We demonstrate the specific phosphorylation of the precursor forms of the chloroplast stroma proteins ferredoxin (preFd), small subunit of ribulose-bisphosphate-carboxylase (preSSU), the thylakoid localized light-harvesting chlorophyll a/b-binding protein (preLHCP), and the thylakoid lumen-localized proteins of the oxygen-evolving complex of 23 kDa (preOE23) and 33 kDa (preOE33). In the case of thylakoid lumen proteins which possess bipartite transit sequences, the phosphorylation occurs within the stroma-targeting domain. By using single amino acid substitution within the presequences of preSSU, preOE23, and preOE33, we were able to tentatively identify a consensus motif for the precursor protein protein kinase. This motif is (P/G)X(n)(R/K)X(n)(S/T)X(n) (S*/T*), were n = 0-3 amino acids spacer and S*/T* represents the phosphate acceptor. The precursor protein protein kinase is present only in plant extracts, e.g. wheat germ and pea, but not in a reticulocyte lysate. Protein import experiments into chloroplasts revealed that phosphorylated preSSU binds to the organelles, but dephosphorylation seems required to complete the translocation process and to obtain complete import. These results suggest that a precursor protein protein phosphatase is involved in chloroplast import and represents a so far unidentified component of the import machinery. In contrast to sucrose synthase, a cytosolic marker protein, the precursor protein protein kinase seems to adhere partially to the chloroplast surface. A phosphorylation-dephosphorylation cycle of chloroplast-destined precursor proteins might represent one step

  6. Simultaneous Kinetic Analysis of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activities 1

    PubMed Central

    Kent, Samuel S.; Young, Joseph D.

    1980-01-01

    An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-14C,5-3H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate Vc/Vo ratios from the expression A/(B-A) where A and B represent the 3H/14C isotope ratios of doubly labeled RuBP and 3-PGA, and Vc and Vo represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-14C]glucose and [6-3H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase. The kinetic properties of a commercial preparation of fully activated spinach carboxylase were studied under approximated physiological conditions of 20% O2 (252 micromolar), 295 μl/l CO2 (10 micromolar), 25 C, and pH 8.19. The Vc/Vo ratio was, within experimental error, constant at 30 seconds and 1 minute. This double label assay method may be used to calculate Vc/Vo ratios for the Laing-Ogren-Hageman equation, Vc/Vo = (VcKo/VoKc) ([CO2]/[O2]) where Vc and Vo represent Vmax, and Kc and Ko represent Michaelis constants for the carboxylase and oxygenase activities, respectively. PMID:16661214

  7. Characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis Z.

    PubMed

    Yokota, A; Harada, A; Kitaoka, S

    1989-03-01

    An improved method was devised to purify ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) with high specific activity (2.1 mumol of CO2 fixed/mg protein/min) from Euglena gracilis Z. The purified enzyme stored at -80 degrees C required treatment with dithiothreitol for full activity. The dithiothreitol-treated RuBisCO was activated by 12 mM NaHCO3 and 20 mM MgCl2, and the activated state was stable at least for 60 min in the presence of 4 mM ethylenediaminetetraacetate. The form of inorganic carbon fixed by the Euglena enzyme was CO2, as for the plant enzymes. The carboxylase reaction proceeded linearly with time for at least 8 min. The optimum pH for this reaction was 7.8 to 8.0. The carboxylase activity increased with increasing temperature up to 50 degrees C. The activation energy for the carboxylation reaction was 10.0 kcal/mol. The Michaelis constants of Euglena RuBisCO were 30.9 microM for CO2, 560 microM for O2, and 10.5 microM for ribulose 1,5-bisphosphate. Mathematical comparison between the photosynthesis rate predicted from these enzymatic properties and the observed rate suggested that there is no CO2-concentrating mechanism in E. gracilis.

  8. Isolation, characterization, and crystallization of ribulosebisphosphate carboxylase from autotrophically grown Rhodospirillum rubrum.

    PubMed

    Schloss, J V; Phares, E F; Long, M V; Norton, I L; Stringer, C D; Hartman, F C

    1979-01-01

    Serial culture of Rhodospirillum rubrum with 2% CO2 in H2 as the exclusive carbon source resulted in a rather large fraction of the soluble protein (greater than 40%) being comprised of ribulosebisphosphate carboxylase (about sixfold higher than the highest value previously reported). Isolation of the enzyme from these cells revealed that it has physical and kinetic properties similar to those previously described for the enzyme derived from cells grown on butyrate. Notably, the small subunit (which is a constituent of the carboxylase from eucaryotes and most procaryotes) was absent in the enzyme from autotrophically grown R. rubrum. Edman degradation of the purified enzyme revealed that the NH2 terminus is free (in contrast to the catalytic subunit of the carboxylase from eucaryotes) and that the NH2-terminal sequence is Met-Asp-Gln-Ser-Ser-Arg-Tyr-Val-Asn-Leu-Ala-Leu-Lys-Glu-Glu-Asp-Leu-Ile-Ala-Gly-Gly-Glx-His-Val-Leu-. Crystals of the enzyme were readily obtained by dialysis against distilled water.

  9. A substrate-induced biotin binding pocket in the carboxyltransferase domain of pyruvate carboxylase.

    PubMed

    Lietzan, Adam D; St Maurice, Martin

    2013-07-05

    Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp(590) and Tyr(628) and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.

  10. Wheat acetyl-coenzyme A carboxylase: cDNA and protein structure.

    PubMed Central

    Gornicki, P; Podkowinski, J; Scappino, L A; DiMaio, J; Ward, E; Haselkorn, R

    1994-01-01

    cDNA fragments encoding part of wheat (Triticum aestivum) acetyl-CoA carboxylase (ACC; EC 6.4.1.2) were cloned by PCR using primers based on the alignment of several biotin-dependent carboxylases. A set of overlapping clones encoding the entire wheat ACC was then isolated by using these fragments as probes. The cDNA sequence contains a 2257-amino acid reading frame encoding a 251-kDa polypeptide. The amino acid sequence of the most highly conserved domain, corresponding to the biotin carboxylases of prokaryotes, is 52-55% identical to ACC of yeast, rat, and diatom. Identity with the available C-terminal amino acid sequence of maize ACC is 66%. The biotin attachment site has the typical eukaryotic EVMKM sequence. The cDNA does not encode an obvious chloroplast targeting sequence. Various cDNA fragments hybridize in Northern blots to a 7.9-kb mRNA. Southern analysis with cDNA probes revealed multiple hybridizing fragments in hexaploid wheat DNA. Some of the wheat cDNA probes also hybridize with ACC-specific DNA from other plants, indicating significant conservation among plant ACCs. Images PMID:7913745

  11. Characterisation and purification of ribulose-bisphosphate carboxylase from heterotrophically grown halophilic archaebacterium, Haloferax mediterranei.

    PubMed

    Rajagopalan, R; Altekar, W

    1994-04-15

    The CO2-fixing enzyme of Calvin cycle ribulose-1,5-bisphosphate-carboxylase/oxygenase has been isolated from a halophilic bacterium, Haloferax mediterranei grown heterotrophically. A homogeneous preparation was obtained from sonicated extract of the cells by three steps, resulting in a specific activity of 52 nmol.min-1.mg protein-1. The physicochemical and catalytic properties of the enzyme were studied. The halobacterial ribulose-bisphosphate carboxylase is an oligomer of 54-kDa and 14-kDa subunits as detected by SDS/PAGE. By sucrose-density-gradient centrifugation, the molecular mass of the enzyme was estimated as approximately 500 kDa indicating a hexadecameric nature. No evidence for an additional form of the enzyme devoid of small subunits was obtained. The enzyme required Mg2+ for activity, KCl for activity and stability, and an optimal pH of 7.8. In contrast to many halophilic proteins, ribulose-bisphosphate carboxylase from H. mediterranei is not an acidic protein. From the comparison of amino acid composition of halobacterial enzyme with its counterparts from a few eukaryotic and eubacterial sources, the S delta Q values showed that these proteins share some compositional similarities.

  12. 3-Methylcrotonyl-CoA carboxylase deficiency: phenotypic variability in a family.

    PubMed

    Eminoglu, F Tuba; Ozcelik, Aysima A; Okur, Ilyas; Tumer, Leyla; Biberoglu, Gursel; Demir, Ercan; Hasanoglu, Alev; Baumgartner, Matthias R

    2009-04-01

    A family with 3-methylcrotonyl-CoA carboxylase deficiency with different clinical features is described. A 15-month-old boy, who was the index patient, was admitted to the hospital with atonic seizure. His brother had delayed language development and their uncle had been followed with diagnosis of epilepsy for the last 5 years. Urinary organic acid analysis displayed elevated 3-hydroxyisovaleric acid and 3-methylcrotonylglycine, analysis of acylcarnitines showed elevated 3-hydroxyisovalerylcarnitine and decreased free carnitine levels in both the patients and their uncle. Methylcrotonyl-CoA carboxylase activity in cultured fibroblasts displayed a low residual activity of 2.2% of the median control value while propionyl-CoA carboxylase activity was normal in the index patient. Mutation analysis revealed a large homozygous deletion of 2264 bp (c.873+4524_6787de12264) in the MCCA gene, which has not been described to date. Adult-onset afebrile seizures have not been reported in the literature. Our cases are an example of this wide phenotypic variability within a single family.

  13. Variations in Kinetic Properties of Ribulose-1,5-bisphosphate Carboxylases among Plants

    PubMed Central

    Yeoh, Hock-Hin; Badger, Murray R.; Watson, Leslie

    1981-01-01

    Studies of ribulose-1,5-bisphosphate (RuBP) carboxylase from taxonomically diverse plants show that the enzyme from C3 and crassulacean acid metabolism pathway species exhibits lower Km(CO2) values (12-25 micromolar) than does that from C4 species (28-34 micromolar). RuBP carboxylase from aquatic angiosperms, an aquatic bryophyte, fresh water and marine algae has yielded consistently high Km(CO2) values (30-70 micromolar), similar in range to that of the enzyme from C4 terrestrial plants. This variation in Km(CO2) is discussed in relation to the correlation between the existence of CO2-concentrating mechanisms for photosynthesis and the affinity of the enzyme for CO2. The Km(RuBP) of the enzyme from various sources ranges from 10 to 136 micromolar; mean ± sd = 36 ± 20 micromolar. This variation in Km(RuBP) does not correlate with different photosynthetic pathways, but shows taxonomic patterns. Among the dicotyledons, the enzyme from crassinucellate species exhibits lower Km(RuBP) (18 ± 4 micromolar) than does that from tenuinucellate species (25 ± 7 micromolar). Among the Poaceae, RuBP carboxylase from Triticeae, chloridoids, andropogonoids, Microlaena, and Tetrarrhena has yielded lower Km(RuBP) values (29 ± 11 micromolar) than has that from other members of the grass family (46 ± 10 micromolar). PMID:16661826

  14. Interplay of light and temperature during the in planta modulation of C4 phosphoenolpyruvate carboxylase from the leaves of Amaranthus hypochondriacus L.: diurnal and seasonal effects manifested at molecular levels.

    PubMed

    Avasthi, Uday K; Izui, Katsura; Raghavendra, Agepati S

    2011-01-01

    The interactive effects of light and temperature on C(4) phosphoenolpyruvate carboxylase (PEPC) were examined both in vivo and in situ using the leaves of Amaranthus hypochondriacus collected at different times during a day and in each month during the year. The maximum activity of PEPC, least inhibition by malate, and highest activation by glucose-6-phosphate were at 15.00 h during a typical day, in all the months. This peak was preceded by maximum ambient light but coincided with high temperature in the field. The highest magnitude in such responses was in the summer (e.g. May) and least in the winter (e.g. December). Light appeared to dominate in modulating the PEPC catalytic activity, whereas temperature had a strong influence on the regulatory properties, suggesting interesting molecular interactions. The molecular mechanisms involved in such interactive effects were determined by examining the PEPC protein/phosphorylation/mRNA levels. A marked diurnal rhythm could be seen in the PEPC protein levels and phosphorylation status during May (summer month). In contrast, only the phosphorylation status increased during the day in December (winter month). The mRNA peaks were not as strong as those of phosphorylation. Thus, the phosphorylation status and the protein levels of PEPC were crucial in modulating the daily and seasonal patterns in C(4) leaves in situ. This is the first detailed study on the diurnal as well as seasonal patterns in PEPC activity, its regulatory properties, protein levels, phosphorylation status, and mRNA levels, in relation to light and temperature intensities in the field.

  15. Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.

    PubMed

    Prihandoko, Rudi; Bradley, Sophie J; Tobin, Andrew B; Butcher, Adrian J

    2015-06-01

    G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype.

  16. Transmembrane domain interactions and residue proline 378 are essential for proper structure, especially disulfide bond formation, in the human vitamin K-dependent gamma-glutamyl carboxylase.

    PubMed

    Tie, Jian-Ke; Zheng, Mei-Yan; Hsiao, Kuang-Ling N; Perera, Lalith; Stafford, Darrel W; Straight, David L

    2008-06-17

    We used recombinant techniques to create a two-chain form (residues 1-345 and residues 346-758) of the vitamin K-dependent gamma-glutamyl carboxylase, a glycoprotein located in the endoplasmic reticulum containing five transmembrane domains. The two-chain carboxylase had carboxylase and epoxidase activities similar to those of one-chain carboxylase. In addition, it had normal affinity for the propeptide of factor IX. We employed this molecule to investigate formation of the one disulfide bond in carboxylase, the transmembrane structure of carboxylase, and the potential interactions among the carboxylase's transmembrane domains. Our results indicate that the two peptides of the two-chain carboxylase are joined by a disulfide bond. Proline 378 is important for the structure necessary for disulfide formation. Results with the P378L carboxylase indicate that noncovalent bonds maintain the two-chain structure even when the disulfide bond is disrupted. As we had previously proposed, the fifth transmembrane domain of carboxylase is the last and only transmembrane domain in the C-terminal peptide of the two-chain carboxylase. We show that the noncovalent association between the two chains of carboxylase involves an interaction between the fifth transmembrane domain and the second transmembrane domain. Results of a homology model of transmembrane domains 2 and 5 suggest that not only do these two domains associate but that transmembrane domain 2 may interact with another transmembrane domain. This latter interaction may be mediated at least in part by a motif of glycine residues in the second transmembrane domain.

  17. Protein phosphorylation in stomatal movement

    PubMed Central

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  18. Phosphorylation site prediction in plants.

    PubMed

    Yao, Qiuming; Schulze, Waltraud X; Xu, Dong

    2015-01-01

    Protein phosphorylation events on serine, threonine, and tyrosine residues are the most pervasive protein covalent bond modifications in plant signaling. Both low and high throughput studies reveal the importance of phosphorylation in plant molecular biology. Although becoming more and more common, the proteome-wide screening on phosphorylation by experiments remains time consuming and costly. Therefore, in silico prediction methods are proposed as a complementary analysis tool to enhance the phosphorylation site identification, develop biological hypothesis, or help experimental design. These methods build statistical models based on the experimental data, and they do not have some of the technical-specific bias, which may have advantage in proteome-wide analysis. More importantly computational methods are very fast and cheap to run, which makes large-scale phosphorylation identifications very practical for any types of biological study. Thus, the phosphorylation prediction tools become more and more popular. In this chapter, we will focus on plant specific phosphorylation site prediction tools, with essential illustration of technical details and application guidelines. We will use Musite, PhosPhAt and PlantPhos as the representative tools. We will present the results on the prediction of the Arabidopsis protein phosphorylation events to give users a general idea of the performance range of the three tools, together with their strengths and limitations. We believe these prediction tools will contribute more and more to the plant phosphorylation research community.

  19. Protein tyrosine phosphorylation in streptomycetes.

    PubMed

    Waters, B; Vujaklija, D; Gold, M R; Davies, J

    1994-07-01

    Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.

  20. Phosphoenol Pyruvate Carboxylase in Parasitic Plants: Further Characterization in Various Species and Localization at the Level of Cells and Tissues in Lathraea clandestina L.

    PubMed

    Renaudin, S; Thalouarn, P; Rey, L; Vidal, J; Larher, F

    1984-11-01

    Phosphoenolpyruvate carboxylase (PEP carboxylase, EC 4.1.1.31) activity was demonstrated in a range of holo and hemiparasitic phanerogams. Lathraea clandestina was used as a model for a more detailed study. Enzyme activity levels were determined in the various plant parts. Great changes in enzyme capacity were observed in the shoots according to the time of measurement during a 24 hr cycle. PEP carboxylase characterized at the cellular level by using an indirect immunofluorescence method was found to be mainly located in the cytosol. The possible functions of PEP carboxylase in parasitic plants are discussed.

  1. Distribution of fallover in the carboxylase reaction and fallover-inducible sites among ribulose 1,5-bisphosphate carboxylase/oxygenases of photosynthetic organisms.

    PubMed

    Uemura, K; Tokai, H; Higuchi, T; Murayama, H; Yamamoto, H; Enomoto, Y; Fujiwara, S; Hamada, J; Yokota, A

    1998-02-01

    The biphasic reaction course, fallover, of carboxylation catalysed by ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been known as a characteristic of the enzyme from higher land plants. Fallover consists of hysteresis in the reaction seen during the initial several minutes and a very slow suicide inhibition by inhibitors formed from the substrate ribulose-1,5-bisphosphate (RuBP). This study examined the relationship between occurrence of fallover and non-catalytic RuBP-binding sites, and the putative hysteresis-inducible sites (Lys-21 and Lys-305 of the large subunit in spinach RuBisCO) amongst RuBisCOs of a wide variety of photosynthetic organisms. Fallover could be detected by following the course of the carboxylase reaction at 1 mM RuBP and the non-catalytic binding sites by alleviation of fallover at 5 mM RuBP. RuBisCO from Euglena gracilis showed the same linear reaction course at both RuBP concentrations, indicating an association between an absence of fallover and an absence of the non-catalytic binding sites. This was supported by the results of an equilibrium binding assay for this enzyme with a transition state analogue. Green macroalgae and non-green algae contained the plant-type, fallover enzyme. RuBisCOs from Conjugatae, Closterium ehrenbergii, Gonatozygon monotaenium and Netrium digitus, showed a much smaller decrease in activity at 1 mM RuBP than the spinach enzyme and the reaction courses of these enzymes at 5 mM RuBP were almost linear. RuBisCO of a primitive type Conjugatae, Mesotaenium caldariorum, showed the same linear course at both RuBP concentrations. Sequencing of rbcL of these organisms indicated that Lys-305 was changed into arginine with Lys-21 conserved.

  2. Cloning and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) cDNA from green microalga Ankistrodesmus convolutus.

    PubMed

    Thanh, Tran; Chi, Vu Thi Quynh; Abdullah, Mohd Puad; Omar, Hishamuddin; Noroozi, Mostafa; Napis, Suhaimi

    2011-11-01

    An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.

  3. Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

    SciTech Connect

    Mochalkin, Igor; Miller, J. Richard; Evdokimov, Artem; Lightle, Sandra; Yan, Chunhong; Stover, Charles Ken; Waldrop, Grover L.

    2008-10-24

    Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or 'flip-flop' their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF{sub 2}P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC.

  4. Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

    PubMed Central

    Mochalkin, Igor; Miller, J. Richard; Evdokimov, Artem; Lightle, Sandra; Yan, Chunhong; Stover, Charles Ken; Waldrop, Grover L.

    2008-01-01

    Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or “flip-flop” their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF2P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC. PMID:18725455

  5. Isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase from leaves.

    PubMed

    Carmo-Silva, A Elizabete; Barta, Csengele; Salvucci, Michael E

    2011-01-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a multifunctional enzyme that catalyzes the fixation of CO2 and O2 in photosynthesis and photorespiration, respectively. As the rate-limiting step in photosynthesis, improving the catalytic properties of Rubisco has long been viewed as a viable strategy for increasing plant productivity. Advances in biotechnology have made this goal more attainable by making it possible to modify Rubisco in planta. To properly evaluate the properties of Rubisco, it is necessary to isolate the enzyme in pure form. This chapter describes procedures for rapid and efficient purification of Rubisco from leaves of several species.

  6. Purification and characterization of the thermostable ribulose-1,5-bisphosphate carboxylase/oxygenase from the thermophilic purple bacterium Chromatium tepidum.

    PubMed

    Heda, G D; Madigan, M T

    1989-09-15

    The Calvin cycle enzyme ribulose-bisphosphate carboxylase/oxygenase has been purified and characterized from the thermophilic and obligately anaerobic purple sulfur bacterium, Chromatium tepidum. The enzyme is an L8S8 carboxylase with a molecular mass near 550 kDa. No evidence for a second form of the enzyme lacking small subunits was obtained. C. tepidum ribulose-bisphosphate carboxylase/oxygenase was stable to heating to temperatures of 60 degrees C and could be readily purified in an active form at room temperature. Both carboxylase and oxygenase activities of this enzyme were Mg2+-dependent and carboxylase activity was sensitive to the effector 6-phosphogluconic acid. The Km for ribulose bisphosphate for the carboxylase activity of the C. tepidum enzyme was substantially higher than that observed in mesophilic Calvin cycle autotrophs. Amino acid composition and immunological analyses of C. tepidum and Chromatium vinosum ribulose-bisphosphate carboxylases showed the enzymes to be highly related despite significant differences in heat stability. It is hypothesized that thermal stability of C. tepidum ribulose-bisphosphate carboxylase/oxygenase is due to differences in primary structure affecting folding patterns in both the large and small subunits and is clearly not the result of any unique quaternary structure of the thermostable enzyme.

  7. A Symmetrical Tetramer for S. aureus Pyruvate Carboxylase in Complex with Coenzyme A

    SciTech Connect

    Yu, L.; Xiang, S; Lasso, G; Gil, D; Valle, M; Tong, L

    2009-01-01

    Pyruvate carboxylase (PC) is a conserved metabolic enzyme with important cellular functions. We report crystallographic and cryo-electron microscopy (EM) studies of Staphylococcus aureus PC (SaPC) in complex with acetyl-CoA, an allosteric activator, and mutagenesis, biochemical, and structural studies of the biotin binding site of its carboxyltransferase (CT) domain. The disease-causing A610T mutation abolishes catalytic activity by blocking biotin binding to the CT active site, and Thr908 might play a catalytic role in the CT reaction. The crystal structure of SaPC in complex with CoA reveals a symmetrical tetramer, with one CoA molecule bound to each monomer, and cryo-EM studies confirm the symmetrical nature of the tetramer. These observations are in sharp contrast to the highly asymmetrical tetramer of Rhizobium etli PC in complex with ethyl-CoA. Our structural information suggests that acetyl-CoA promotes a conformation for the dimer of the biotin carboxylase domain of PC that might be catalytically more competent.

  8. Dark/Light Modulation of Ribulose Bisphosphate Carboxylase Activity in Plants from Different Photosynthetic Categories 1

    PubMed Central

    Vu, J. Cu V.; Allen, Leon H.; Bowes, George

    1984-01-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO3− and Mg2+ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C3); P. maximum (C4 phosphoenolpyruvate carboxykinase); P. milioides (C3/C4); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C3); P. miliaceum (C4 NAD malic enzyme); Zea mays and Sorghum bicolor (C4 NADP malic enzyme); Moricandia arvensis (C3/C4); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C3 species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO2 and Mg2+ activation, but which can be converted to an activatable state upon exposure of the leaf to light. PMID:16663937

  9. Dark/Light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories.

    PubMed

    Vu, J C; Allen, L H; Bowes, G

    1984-11-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO(3) (-) and Mg(2+) concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C(3)); P. maximum (C(4) phosphoenolpyruvate carboxykinase); P. milioides (C(3)/C(4)); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C(3)); P. miliaceum (C(4) NAD malic enzyme); Zea mays and Sorghum bicolor (C(4) NADP malic enzyme); Moricandia arvensis (C(3)/C(4)); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C(3) species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO(2) and Mg(2+) activation, but which can be converted to an activatable state upon exposure of the leaf to light.

  10. Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

    PubMed

    Houtz, R L; Stults, J T; Mulligan, R M; Tolbert, N E

    1989-03-01

    Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of N epsilon-trimethylation.

  11. The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase.

    PubMed

    Lietzan, Adam D; Lin, Yi; St Maurice, Martin

    2014-11-15

    Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes.

  12. Interaction of ribulose bisphosphate carboxylase/oxygenase with 2-carboxyhexitol 1,6-bisphosphates.

    PubMed

    Roach, D J; Gollnick, P D; McFadden, B A

    1983-04-01

    2-C-Carboxy-D-glucitol 1,6-bisphosphate (CGBP) and 2-C-carboxy-D-mannitol 1,6-bisphosphate (CMBP) have been synthesized, isolated, and the structures of these compounds and the derived lactones elucidated by NMR spectroscopy and periodate oxidation. Both carboxyhexitol bisphosphates, which are homologs of the transition state analog 2-C-carboxy-D-arabinitol 1,5-bisphosphate, exhibit competitive inhibiton of ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.9) isolated from spinach (Spinacia oleracea), with respect to ribulose 1,5-bisphosphate. CMBP was a more potent inhibitor (100-fold) displaying an inhibition constant (Ki at pH 8.0 and 30 degrees C) of 1-2 microM with enzymes from spinach, barley (Hordeum vulgare), and Chromatium vinosum. In contrast the Rhodospirillum rubrum enzyme was inhibited about 40-fold more weakly (Ki = 53 microM at pH 8.0 and 30 degrees C). Both CGBP and CMBP potentiated activation of RuBP carboxylase from spinach and R. rubrum.

  13. Dark/light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories

    SciTech Connect

    Vu, J.C.V.; Allen, L.H. Jr.; Bowes, G.

    1984-11-01

    Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from light-exposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO/sub 3//sup -/ and Mg/sup 2 +/ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C/sub 3/); P. maximum (C/sub 4/ phosphoenolpyruvate carboxykinase); P. milioides (C/sub 3//C/sub 4/); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C/sub 3/); P. miliaceum (C/sub 4/ NAD malic enzyme); Zea mays and Sorghum bicolor (C/sub 4/ NADP malic enzyme); Moricandia arvensis (C/sub 3//C/sub 4/); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C/sub 3/ species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO/sub 2/ and Mg/sup 2 +/ activation, but which can be converted to an activatable state upon exposure of the leaf to light. 16 references, 2 tables.

  14. Discovery of Antibacterial Biotin Carboxylase Inhibitors by Virtual Screening and Fragment-Based Approaches

    SciTech Connect

    Mochalkin, Igor; Miller, J. Richard; Narasimhan, Lakshmi; Thanabal, Venkataraman; Erdman, Paul; Cox, Philip B.; Prasad, J.V.N. Vara; Lightle, Sandra; Huband, Michael D.; Stover, C. Kendall; Pfizer

    2009-07-24

    As part of our effort to inhibit bacterial fatty acid biosynthesis through the recently validated target biotin carboxylase, we employed a unique combination of two emergent lead discovery strategies. We used both de novo fragment-based drug discovery and virtual screening, which employs 3D shape and electrostatic property similarity searching. We screened a collection of unbiased low-molecular-weight molecules and identified a structurally diverse collection of weak-binding but ligand-efficient fragments as potential building blocks for biotin carboxylase ATP-competitive inhibitors. Through iterative cycles of structure-based drug design relying on successive fragment costructures, we improved the potency of the initial hits by up to 3000-fold while maintaining their ligand-efficiency and desirable physicochemical properties. In one example, hit-expansion efforts resulted in a series of amino-oxazoles with antibacterial activity. These results successfully demonstrate that virtual screening approaches can substantially augment fragment-based screening approaches to identify novel antibacterial agents.

  15. Translational regulation of light-induced ribulose 1,5-bisphosphate carboxylase gene expression in amaranth.

    PubMed Central

    Berry, J O; Nikolau, B J; Carr, J P; Klessig, D F

    1986-01-01

    The regulation of the genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase was examined in amaranth cotyledons in response to changes in illumination. When dark-grown cotyledons were transferred into light, synthesis of the large- and small-subunit polypeptides was initiated very rapidly, before any increase in the levels of their corresponding mRNAs. Similarly, when light-grown cotyledons were transferred to total darkness, synthesis of the large- and small-subunit proteins was rapidly depressed without changes in mRNA levels for either subunit. In vitro translation or in vivo pulse-chase experiments indicated that these apparent changes in protein synthesis were not due to alterations in the functionality of the mRNAs or to protein turnover, respectively. These results, in combination with our previous studies, suggest that the expression of ribulose 1,5-bisphosphate carboxylase genes can be adjusted rapidly at the translational level and over a longer period through changes in mRNA accumulation. Images PMID:3785198

  16. Light-mediated control of translational initiation of ribulose-1, 5-bisphosphate carboxylase in amaranth cotyledons.

    PubMed Central

    Berry, J O; Breiding, D E; Klessig, D F

    1990-01-01

    In cotyledons of 6-day-old amaranth seedlings, the large subunit (LSU) and the small subunit (SSU) polypeptides of ribulose-1,5-bisphosphate carboxylase are not synthesized in the absence of light. When dark-grown seedlings were transferred into light, synthesis of both polypeptides was induced within the first 3 to 5 hr of illumination without any significant changes in levels of their mRNAs. In cotyledons of light-grown seedlings and of dark-grown seedlings transferred into light for 5 hr (where ribulose-1,5-bisphosphate carboxylase synthesis was readily detected in vivo), the LSU and SSU mRNAs were associated with polysomes. In cotyledons of dark-grown seedlings, these two mRNAs were not found on polysomes. In contrast to the SSU message, mRNAs encoding the nonlight-regulated, nuclear-encoded proteins actin and ubiquitin were associated with polysomes regardless of the light conditions. Similarly, mRNA from at least one chloroplast-encoded gene (rpl2) was found on polysomes in the dark as well as in the light. These results indicate an absence of translational initiation in cotyledons of dark-grown seedlings which is specific to a subset of nuclear- and chloroplast-encoded genes including the SSU and LSU, respectively. Upon illumination, synthesis of both polypeptides, and possibly other proteins involved in light-mediated chloroplast development, was induced at the level of translational initiation. PMID:2152128

  17. Active site histidine in spinach ribulosebisphosphate carboxylase/oxygenase modified by diethyl pyrocarbonate

    SciTech Connect

    Igarashi, Y.; McFadden, B.A.; el-Gul, T.

    1985-07-16

    (TH) Diethyl pyrocarbonate was synthesized from (TH) ethanol prepared by the reduction of acetaldehyde by NaB3H4. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach was inactivated with this reagent at pH 7.0 the presence of 20 mM MgS , and tryptic peptides that contained modified histidine residues were isolated by reverse-phase high-performance liquid chromatography. Labeling of the enzyme was conducted in the presence and absence of the competitive inhibitor sedoheptulose 1,7-bisphosphate. The amount of one peptide that was heavily labeled in the absence of this compound was reduced 10-fold in its presence. The labeled residue was histidine-298. This result, in combination with earlier experiments, suggests that His-298 in spinach RuBisCO is located in the active site domain and is essential to enzyme activity. This region of the primary structure is strongly conserved in seven other ribulosebisphosphate carboxylases from divergent sources.

  18. Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from Anabaena 7210 expressed in Escherichia coli

    SciTech Connect

    Gurevitz, M.; Somerville, C.R.; McIntosh, L.

    1985-10-01

    The authors have placed the genes encoding ribulosebisphosphate carboxylase/oxygenase from the Anabaena 7120 operon under transcriptional control of the lac promoter carried on the Escherichia coli plasmid pUC19. The genes encoding both the large and small subunit polypeptides (rbcL and rbcS) are transcribed and translated so that approx. = 0.6% of the soluble protein in E. coli extracts is a fully functional holoenzyme with a sedimentation coefficient of approximately 18S, which contains stoichiometric amounts of the two subunits. However, expression of the large subunit polypeptide vastly exceeds that of the small subunit because the majority of transcripts terminate in the intergenic region between the rbcL and rbcS genes. As a result, excess large subunit is synthesized and accumulates in E. coli as an insoluble and catalytically inactive form. Because small subunit is found only in the high molecular weight soluble form of ribulosebisphosphate carboxylase/oxygenase, the authors propose that the small subunit promotes assembly of the hexadecameric form of the enzyme via heterodimers of large and small subunits.

  19. N-->S phosphoryl migration in phosphoryl glutathion.

    PubMed

    Yang, H J; Liu, J; Zhao, Y F

    1993-07-01

    It was found that in the case of N-(diisopropylphosphoryl) glutathion (reduced form), 2, N-->S phosphoryl migration took place, but not for N,N-bis(diisopropylphosphoryl) glutathion (oxidized form) or N-diisopropylphosphoryl cysteine. These results were deduced by 31P-NMR tracing experiments. It was shown that phosphoryl migration was catalyzed by an intramolecular carboxyl group, and a mechanism involving a mixed carboxyl-phosphoric anhydride was proposed. A competitive reaction between the amino and thiol group toward diisopropyl phosphite indicated that the phospho-thiol derived from N-(diisopropylphosphoryl) glutathion (reduced form), 2, did not result from direct phosphorylation of the thiol group. N,S-Bis(diisopropylphosphoryl) glutathion provides an authentic sample to confirm the migrated phosphoryl thiol product.

  20. Oxidative and Photosynthetic Phosphorylation Mechanisms

    ERIC Educational Resources Information Center

    Wang, Jui H.

    1970-01-01

    Proposes a molecular mechanism for the coupling of phosphorylation to electron transport in both mitochondria and chloroplasts. Justifies the proposed reaction schemes in terms of thermodynamics and biochemical data. Suggests how areobic respiration could have evolved. (EB)

  1. Phosphorylation regulates human OCT4.

    PubMed

    Brumbaugh, Justin; Hou, Zhonggang; Russell, Jason D; Howden, Sara E; Yu, Pengzhi; Ledvina, Aaron R; Coon, Joshua J; Thomson, James A

    2012-05-08

    The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.

  2. Phosphorylation regulates human OCT4

    PubMed Central

    Brumbaugh, Justin; Russell, Jason D.; Howden, Sara E.; Yu, Pengzhi; Ledvina, Aaron R.; Coon, Joshua J.; Thomson, James A.

    2012-01-01

    The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography–MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency. PMID:22474382

  3. Expression, purification, and characterization of human acetyl-CoA carboxylase 2.

    PubMed

    Kim, Ki Won; Yamane, Harvey; Zondlo, James; Busby, James; Wang, Minghan

    2007-05-01

    The full-length human acetyl-CoA carboxylase 1 (ACC1) was expressed and purified to homogeneity by two separate groups (Y.G. Gu, M. Weitzberg, R.F. Clark, X. Xu, Q. Li, T. Zhang, T.M. Hansen, G. Liu, Z. Xin, X. Wang, T. McNally, H. Camp, B.A. Beutel, H.I. Sham, Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors, J. Med. Chem. 49 (2006) 3770-3773; D. Cheng, C.H. Chu, L. Chen, J.N. Feder, G.A. Mintier, Y. Wu, J.W. Cook, M.R. Harpel, G.A. Locke, Y. An, J.K. Tamura, Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes, Protein Expr. Purif., in press). However, neither group was successful in expressing the full-length ACC2 due to issues of solubility and expression levels. The two versions of recombinant human ACC2 in these reports are either truncated (lacking 1-148 aa) or have the N-terminal 275 aa replaced with the corresponding ACC1 region (1-133 aa). Despite the fact that ACC activity was observed in both cases, these constructs are not ideal because the N-terminal region of ACC2 could be important for the correct folding of the catalytic domains. Here, we report the high level expression and purification of full-length human ACC2 that lacks only the N-terminal membrane attachment sequence (1-20 and 1-26 aa, respectively) in Trichoplusia ni cells. In addition, we developed a sensitive HPLC assay to analyze the kinetic parameters of the recombinant enzyme. The recombinant enzyme is a soluble protein and has a K(m) value of 2 microM for acetyl-CoA, almost 30-fold lower than that reported for the truncated human ACC2. Our recombinant enzyme also has a lower K(m) value for ATP (K(m)=52 microM). Although this difference could be ascribed to different assay conditions, our data suggest that the longer human ACC2 produced in our system may have higher affinities for the substrates and could

  4. Isolation and preliminary characterization of two forms of ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas capsulata.

    PubMed

    Gibson, J L; Tabita, F R

    1977-12-01

    The presence of two distinct forms of ribulose 1,5-bisphosphate carboxylase has been demonstrated in extracts of Rhodopseudomonas capsulata, similar to the form I (peak I) and form II (peak II) carboxylases previously described from R. sphaeroides (J. Gibson and F. R. Tabita, J. Biol. Chem 252:943-949, 1977). The two activities, separated by diethylaminoethyl-cellulose chromatography, were shown to be of different molecular size after assay on polyacrylamide gels. The higher-molecular-weight carboxylase from R. capsulata was designated form I-C, whereas the smaller enzyme was designated form II-C. Catalytic studies revealed significant differences between the two enzymes in response to pH and the effector 6-phosphogluconate. Immunological studies with antisera directed against the carboxylases from R. sphaeroides demonstrated antigenic differences between the two R. capsulata enzymes; cross-reactivity was observed only between R. sphaeroides anti-form II serum and the corresponding R. capsulata enzyme, form II-C.

  5. Species Variation in the Predawn Inhibition of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase 1

    PubMed Central

    Servaites, Jerome C.; Parry, Martin A. J.; Gutteridge, Steven; Keys, Alfred J.

    1986-01-01

    The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured in extracts of leaves collected before dawn (predawn activity, pa) and at midday (midday activity, ma). Twenty-three of the 37 species examined showed a pa/ma ratio (≤0.75, while only Capsicum frutescens, Cucumis sativa, Glycine max, Nicotiana tabacum, Vigna unguiculata, and 3 Solanum species showed a pa/ma ratio ≤0.5. Phaseolus vulgaris consistently showed a pa/ma ratio of ≤0.1. Activities and pa/ma ratios of the same species grown in the United States and the United Kingdom were very similar. Gel filtration of extracts before assay had no effect on the observed activities and the pa/ma ratios. These data are consistent with the hypothesis that in a number of species the enzyme is partially inhibited following the night period by the presence of a tight-binding inhibitor. PMID:16665155

  6. Purification and characterization of ribulose-1,5-bisphosphate carboxylase from triticale.

    PubMed

    Khan, M A; Dixit, A; Upadhyaya, K C

    1994-04-01

    Ribulose-1,5-bisphosphate carboxylase has been isolated from a synthetic cereal triticale and purified using a newly developed rapid procedure involving precipitation with ammonium sulphate (35-55% saturation), DEAE-cellulose (DE-52) chromatography and filtration through Sepharose CL-68. Molecular weights of the enzyme subunits are 15.5 and 52 kDa which corresponds to 540 kDa for the hexadecameric holoenzyme. Isoelectric focussing showed that the enzyme has a pI of 4.2. Various kinetic constants determined under aerobic conditions are: Km (CO2), 118 microM; Km (RuBP), 220 microM (at 20 mM NaHCO3) and Vmax, 690 nmole CO2 fixed/mg enzyme/min.

  7. Cloning and characterization of ribulose bisphosphate carboxylase gene of a carboxydobacterium, hydrogenophagea pseudoflava DSM 1084.

    PubMed

    Lee, S N; Kim, Y M

    1998-10-31

    The ribulose bisphosphate carboxylase/oxygenase rbcL and rbcS genes of a carbon monoxide-oxidizing bacterium, Hydrogenophaga pseudoflava DSM 1084, were cloned and sequenced. The cloned rbcL and rbcS genes had open reading frames of 1422 and 351 nucleotides encoding RbcL and RbcS with calculated molecular masses of 52,689 and 13,541, respectively. The known active site residues in other RbcL proteins were conserved in the H. pseudoflava proteins. The H. pseudoflava RbcS protein lacked the 12-residue internal sequence found in the plant enzymes. The 2 genes were separated by a 134 bp intergenic region and cotranscribed as a 2.0 kb rbcLS mRNA. Novel two perfect 9 bp direct repeats overlapping with two dyad symmetries were found in the rbcLS promoter region.

  8. Postimport methylation of the small subunit of ribulose-1,5-bisphosphate carboxylase in chloroplasts.

    PubMed

    Grimm, R; Grimm, M; Eckerskorn, C; Pohlmeyer, K; Röhl, T; Soll, J

    1997-05-26

    Electron impact mass spectronomy analysis of the amino-terminal amino acid of the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco) showed that the amino-terminal methionine residue is post-translationally modified to N-methyl-methionine. Modification of the amino-terminal methionine residue was found in mature SSU proteins from the dicotyledonous plants pea and spinach as well as the monocotyledonous plants barley and corn. SSU methyltransferase is a soluble protein in the chloroplast stroma and accepts heterologously expressed non-methylated SSU as a substrate using S-adenosylmethionine as methyl-group donor. We show that this modification occurs after post-translational uptake of the precursor form of SSU into chloroplasts and processing to its mature size. This reaction represents a new step in the import and assembly pathway of Rubisco holoenzyme.

  9. Maternal 3-methylcrotonyl-coenzyme A carboxylase deficiency with elevated 3-hydroxyisovalerylcarnitine in breast milk

    PubMed Central

    Cho, Kyung Lae; Kim, Yeo Jin; Yang, Song Hyun; Kim, Gu-Hwan

    2016-01-01

    We report here a case of maternal 3-methylcrotonyl-coenzyme A carboxylase (3-MCC) deficiency in a Korean woman. Her 2 infants had elevated 3-hydroxyisovalerylcarnitine (C5-OH) on a neonatal screening test by liquid chromatography-tandem mass spectrometry (LC-MS/MS), but normal results were found on urine organic acid analysis. The patient was subjected to serial testing and we confirmed a maternal 3-MCC deficiency by blood spot and breast milk spot test by LC-MS/MS, serum amino acid analysis, urine organic acid and molecular genetic analysis that found c.838G>T (p.Asp280Tyr) homozygous mutation within exon 9 of the MCCB gene. Especially, we confirmed marked higher levels of C5-OH on breast milk spot by LC-MS/MS, in the case of maternal 3-MCC deficiency vs. controls. PMID:28018443

  10. Acetyl-CoA carboxylase inhibitors from avocado (Persea americana Mill) fruits.

    PubMed

    Hashimura, H; Ueda, C; Kawabata, J; Kasai, T

    2001-07-01

    A methanol extract of avocado fruits showed potent inhibitory activity against acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. The active principles were isolated and identified as (5E,12Z,15Z)-2-hydroxy-4-oxoheneicosa-5,12,15-trienyl (1), (2R,12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dienyl (2), (2R*,4R*)-2,4-dihydroxyheptadec-16-enyl (3) and (2R*,4R*)-2,4-dihydroxyheptadec-16-ynyl (4) acetates by instrumental analyses. The IC50 of the compounds were 4.0 x 10(-6), 4.9 x 10(-6), 9.4 x 10(-6), and 5.1 x 10(-6) M, respectively.

  11. [Functions of plant phosphoenolpyruvate carboxylase and its applications for genetic engineering].

    PubMed

    Wei, Shaowei; Li, Yin

    2011-12-01

    Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is an important ubiquitous cytosol enzyme that fixes HCO3 together with phosphoenolpyruvate (PEP) and yields oxaloacetate that can be converted to intermediates of the citric acid cycle. In plant cells, PEPC participates in CO2 assimilation and other important metabolic pathways, and it has broad functions in different plant tissues. PEPC is also involved in the regulation of storage product synthesis and metabolism in seeds, such as affecting the metabolic fluxes from sugars/starch towards the synthesis of fatty acids or amino acids and proteins. In this review, we introduced the progress in classification, structure and regulation of PEPC in plant tissues. We discussed the potential applications of plant PEPCs in genetic engineering. The researches in functions and regulation mechanism of plant PEPCs will provide beneficial approaches to applications of plant PEPCs in high-yield crops breeding, energy crop and microbe genetic engineering.

  12. Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

    SciTech Connect

    Chou, Chi-Yuan; Tong, Liang

    2012-06-19

    Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

  13. Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

    SciTech Connect

    C Chou; L Tong

    2011-12-31

    Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

  14. Ribulose 1,5-bisphosphate carboxylase from the halophilic cyanobacterium Aphanothece halophytica.

    PubMed

    Asami, S; Takabe, T; Akazawa, T; Codd, G A

    1983-09-01

    Various structural and functional properties of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isolated from the halophilic cyanobacterium (blue-green alga) Aphanothece halophytica were reexamined. The ready dissociation of this algal RuBisCO during sedimentation in a linear sucrose density gradient was observed. Low NaCl concentrations promote the dissociation of small subunit (B) from the original native enzyme molecule as evidenced by the sucrose density gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is thus possible that the intracellular osmoticum of A. halophytica might influence the structural integrity and activity of RuBisCO. The low residual carboxylase activity ascribed to the catalytic core, an oligomer form of the large subunit (A) apparently deficient in small subunit (B), was found to be markedly stimulated by a protein component which appears identical to subunit B. The purification and structural characterization of the catalytic core and subunit B were attempted by step-wise column chromatography on DEAE-cellulose, Utrogel AcA 34, Sephadex G-75, and hydroxylapatite, and at the final stage each component was purified to near homogeneity, although the catalytic core is still associated with a small quantity of subunit B. The addition of subunit B to the catalytic core does not alter the Km (HCO-3, RuBP) values, but Vmax values are markedly enhanced. Sucrose density gradient centrifugation gave a value of 16 S for the catalytic core. The molecular weights of the monomeric forms of the catalytic core (subunit A) and subunit B were 5.0 X 10(4) and 1.4 X 10(4), respectively.

  15. Crystal Structure of Biotin Carboxylase in Complex with Substrates and Implications for Its Catalytic Mechanism

    SciTech Connect

    Chou, C.; Yu, L; Tong, L

    2009-01-01

    Biotin-dependent carboxylases are widely distributed in nature and have important functions in many cellular processes. These enzymes share a conserved biotin carboxylase (BC) component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the donor. Despite the availability of a large amount of biochemical and structural information on BC, the molecular basis for its catalysis is currently still poorly understood. We report here the crystal structure at 2.0 {angstrom} resolution of wild-type Escherichia coli BC in complex with its substrates biotin, bicarbonate, and Mg-ADP. The structure suggests that Glu{sup 296} is the general base that extracts the proton from bicarbonate, and Arg{sup 338} is the residue that stabilizes the enolate biotin intermediate in the carboxylation reaction. The B domain of BC is positioned closer to the active site, leading to a 2-{angstrom} shift in the bound position of the adenine nucleotide and bringing it near the bicarbonate for catalysis. One of the oxygen atoms of bicarbonate is located in the correct position to initiate the nucleophilic attack on ATP to form the carboxyphosphate intermediate. This oxygen is also located close to the N1' atom of biotin, providing strong evidence that the phosphate group, derived from decomposition of carboxyphosphate, is the general base that extracts the proton on this N1' atom. The structural observations are supported by mutagenesis and kinetic studies. Overall, this first structure of BC in complex with substrates offers unprecedented insights into the molecular mechanism for the catalysis by this family of enzymes.

  16. Ribulose 1,5-bisphosphate carboxylase/oxygenase from Pseudomonas oxalacticus.

    PubMed Central

    Lawlis, V B; Gordon, G L; McFadden, B A

    1979-01-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase was purified by a rapid, facile procedure from formate-grown Pseudomonas oxalaticus. The electrophoretically homogeneous enzyme had specific activities of 1.9 mumol of CO2 fixed per min per mg of protein and 0.15 mumol of O2 consumed per min per mg of protein. The amino acid composition was similar to that of other bacterial sources of the enzyme. The molecular weights determined by sedimentation equilibrium and by gel filtration were 421,000 and 450,000, respectively. Upon sodium dodecyl sulfate electrophoresis of enzyme purified under conditions which would limit proteolysis, two types of large (L) subunits and two types of small (S) subunits were observed with apparent molecular weights of 57,000, 55,000, 17,000 and 15,000. By densitometric scans at two different protein concentrations the stoichiometry of the total large to total small subunits was 1:1, implying an L6S6 structure. Electron micrographs of the enzyme revealed an unusual structure that was inconsistent with a cubical structure. The enzyme had an unusually high Km for ribulose 1,5-bisphosphate (220 microM) and was strongly inhibited by 6-phosphogluconate in the ribulose 1,5-bisphosphate carboxylase assay (Ki = 270 microM). One, 5, and 12 days after purification the enzyme was half-maximally activated at 0.13 microM, 0.23 mM, and 0.70 mM CO2, respectively, at saturating Mg2+. At saturating CO2, enzyme 1 day afer purification responded sigmoidally to Mg2+ and was half-maximally activated by 0.85 mM Mg2+ in the absence of 6-phosphogluconate (Hill coefficient, h = 2.0) and by 0.19 mM Mg2+ in the presence of mM 6-phosphogluconate (h = 1.7). Images PMID:457602

  17. Nitric oxide regulation of leaf phosphoenolpyruvate carboxylase-kinase activity: implication in sorghum responses to salinity.

    PubMed

    Monreal, José A; Arias-Baldrich, Cirenia; Tossi, Vanesa; Feria, Ana B; Rubio-Casal, Alfredo; García-Mata, Carlos; Lamattina, Lorenzo; García-Mauriño, Sofía

    2013-11-01

    Nitric oxide (NO) is a signaling molecule that mediates many plant responses to biotic and abiotic stresses, including salt stress. Interestingly, salinity increases NO production selectively in mesophyll cells of sorghum leaves, where photosynthetic C₄ phosphoenolpyruvate carboxylase (C₄ PEPCase) is located. PEPCase is regulated by a phosphoenolpyruvate carboxylase-kinase (PEPCase-k), which levels are greatly enhanced by salinity in sorghum. This work investigated whether NO is involved in this effect. NO donors (SNP, SNAP), the inhibitor of NO synthesis NNA, and the NO scavenger cPTIO were used for long- and short-term treatments. Long-term treatments had multifaceted consequences on both PPCK gene expression and PEPCase-k activity, and they also decreased photosynthetic gas-exchange parameters and plant growth. Nonetheless, it could be observed that SNP increased PEPCase-k activity, resembling salinity effect. Short-term treatments with NO donors, which did not change photosynthetic gas-exchange parameters and PPCK gene expression, increased PEPCase-k activity both in illuminated leaves and in leaves kept at dark. At least in part, these effects were independent on protein synthesis. PEPCase-k activity was not decreased by short-term treatment with cycloheximide in NaCl-treated plants; on the contrary, it was decreased by cPTIO. In summary, NO donors mimicked salt effect on PEPCase-k activity, and scavenging of NO abolished it. Collectively, these results indicate that NO is involved in the complex control of PEPCase-k activity, and it may mediate some of the plant responses to salinity.

  18. Methylation of γ-carboxylated Glu (Gla) allows detection by liquid chromatography-mass spectrometry and the identification of Gla residues in the γ-glutamyl carboxylase.

    PubMed

    Hallgren, K W; Zhang, D; Kinter, M; Willard, B; Berkner, K L

    2013-06-07

    γ-Carboxylated Glu (Gla) is a post-translational modification required for the activity of vitamin K-dependent (VKD) proteins that has been difficult to study by mass spectrometry due to the properties of this negatively charged residue. Gla is generated by a single enzyme, the γ-glutamyl carboxylase, which has broad biological impact because VKD proteins have diverse functions that include hemostasis, apoptosis, and growth control. The carboxylase also contains Glas, of unknown function, and is an integral membrane protein with poor sequence coverage. To locate these Glas, we first established methods that resulted in high coverage (92%) of uncarboxylated carboxylase. Subsequent analysis of carboxylated carboxylase identified a Gla peptide (729-758) and a missing region (625-647) that was detected in uncarboxylated carboxylase. We therefore developed an approach to methylate Gla, which efficiently neutralized Gla and improved mass spectrometric analysis. Methylation eliminated CO2 loss from Gla, increased the ionization of Gla-containing peptide, and appeared to facilitate trypsin digestion. Methylation of a carboxylated carboxylase tryptic digest identified Glas in the 625-647 peptide. These studies provide valuable information for testing the function of carboxylase carboxylation. The methylation approach for studying Gla by mass spectrometry is an important advance that will be broadly applicable to analyzing other VKD proteins.

  19. Methylation of gamma-carboxylated Glu (Gla) allows detection by liquid chromatography-mass spectrometry and the identification of Gla residues in the gamma-glutamyl carboxylase

    PubMed Central

    Hallgren, K. W.; Zhang, D.; Kinter, M.; Willard, B.; Berkner, K. L.

    2013-01-01

    Gamma-carboxylated Glu (Gla) is a post-translational modification required for the activity of vitamin K-dependent (VKD) proteins that has been difficult to study by mass spectrometry due to the properties of this negatively-charged residue. Gla is generated by a single enzyme, the gamma-glutamyl carboxylase, which has broad biological impact because VKD proteins have diverse functions that include hemostasis, apoptosis, and growth control. The carboxylase also contains Glas, of unknown function, and is an integral membrane protein with poor sequence coverage. To locate these Glas, we first established methods that resulted in high coverage (92%) of uncarboxylated carboxylase. Subsequent analysis of carboxylated carboxylase identified a Gla-peptide (729-758) and a missing region (625-647) that was detected in uncarboxylated carboxylase. We therefore developed an approach to methylate Gla, which efficiently neutralized Gla and improved mass spectrometric analysis. Methylation eliminated CO2 loss from Gla, increased the ionization of Gla-containing peptide, and appeared to facilitate trypsin digestion. Methylation of a carboxylated carboxylase tryptic digest identified Glas in the 625-647 peptide. These studies provide valuable information for testing the function of carboxylase carboxylation. The methylation approach for studying Gla by mass spectrometry is an important advance that will be broadly applicable to analyzing other VKD proteins. PMID:22536908

  20. Genes encoding the biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution.

    PubMed

    Li, Zhi-Guo; Yin, Wei-Bo; Song, Li-Ying; Chen, Yu-Hong; Guan, Rong-Zhan; Wang, Jing-Qiao; Wang, Richard R-C; Hu, Zan-Min

    2011-03-01

    Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric acetyl-CoA carboxylase (ACCase) that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin carboxyl carrier protein, and CO2 to form carboxybiotin carboxyl carrier protein. In this study, we cloned four genes encoding BC from Brassica napus L. (namely BnaC.BC.a, BnaC.BC.b, BnaA.BC.a, and BnaA.BC.b), and two were cloned from each of the two parental species Brassica rapa L. (BraA.BC.a and BraA.BC.b) and Brassica oleracea L. (BolC.BC.a and BolC.BC.b). Sequence analyses revealed that in B. napus the genes BnaC.BC.a and BnaC.BC.b were from the C genome of B. oleracea, whereas BnaA.BC.a and BnaA.BC.b were from the A genome of B. rapa. Comparative and cluster analysis indicated that these genes were divided into two major groups, BnaC.BC.a, BnaA.BC.a, BraA.BC.a, and BolC.BC.a in group-1 and BnaC.BC.b, BnaA.BC.b, BraA.BC.b, and BolC.BC.b in group-2. The divergence of group-1 and group-2 genes occurred in their common ancestor 13-17 million years ago (MYA), soon after the divergence of Arabidopsis and Brassica (15-20 MYA). This time of divergence is identical to the previously reported triplicated time of paralogous subgenomes of diploid Brassica species and the divergence date of group-1 and group-2 genes of α-carboxyltransferase, another subunit of heteromeric ACCase, in Brassica. Reverse transcription PCR revealed that the expression level of group-1 and group-2 genes varied in different organs, and the expression patterns of the two groups of genes were similar in different organs, except in flower. However, two paralogs of group-2 BC genes from B. napus could express differently in mature plants tested by generating BnaA.BC.b and BnaC.BC.b promoter-β-glucuronidase (GUS) fusions. The amino acid sequences of proteins encoded by these genes were highly conserved, except the sequence encoding

  1. Species having C4 single-cell-type photosynthesis in the Chenopodiaceae family evolved a photosynthetic phosphoenolpyruvate carboxylase like that of Kranz-type C4 species.

    PubMed

    Lara, María Valeria; Chuong, Simon D X; Akhani, Hossein; Andreo, Carlos Santiago; Edwards, Gerald E

    2006-10-01

    Spatial and temporal regulation of phosphoenolpyruvate carboxylase (PEPC) is critical to the function of C(4) photosynthesis. The photosynthetic isoform of PEPC in the cytosol of mesophyll cells in Kranz-type C(4) photosynthesis has distinctive kinetic and regulatory properties. Some species in the Chenopodiaceae family perform C(4) photosynthesis without Kranz anatomy by spatial separation of initial fixation of atmospheric CO(2) via PEPC from C(4) acid decarboxylation and CO(2) donation to Rubisco within individual chlorenchyma cells. We studied molecular and functional features of PEPC in two single-cell functioning C(4) species (Bienertia sinuspersici, Suaeda aralocaspica) as compared to Kranz type (Haloxylon persicum, Salsola richteri, Suaeda eltonica) and C(3) (Suaeda linifolia) chenopods. It was found that PEPC from both types of C(4) chenopods displays higher specific activity than that of the C(3) species and shows kinetic and regulatory characteristics similar to those of C(4) species in other families in that they are subject to light/dark regulation by phosphorylation and display differential malate sensitivity. Also, the deduced amino acid sequence from leaf cDNA indicates that the single-cell functioning C(4) species possesses a Kranz-type C(4) isoform with a Ser in the amino terminal. A phylogeny of PEPC shows that isoforms in the two single-cell functioning C(4) species are in a clade with the C(3) and Kranz C(4) Suaeda spp. with high sequence homology. Overall, this study indicates that B. sinuspersici and S. aralocaspica have a C(4)-type PEPC similar to that in Kranz C(4) plants, which likely is required for effective function of C(4) photosynthesis.

  2. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    SciTech Connect

    JOHN C WALKER

    2011-11-01

    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  3. Phosphorylation and dephosphorylation of spectrin.

    PubMed

    Fairbanks, G; Avruch, J; Dino, J E; Patel, V P

    1978-01-01

    The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of 32Pi from [32P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [32P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions--notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated 1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and 2) by membrane deformation that alters enzyme-spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to

  4. Phosphorylation in halobacterial signal transduction.

    PubMed Central

    Rudolph, J; Tolliday, N; Schmitt, C; Schuster, S C; Oesterhelt, D

    1995-01-01

    Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya. Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world. Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides. The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively. The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation. Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of [gamma-32P]ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled. Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY. The mutant CheY(D53A) had no effect on phosphorylated CheA. The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli. Images PMID:7556066

  5. Effects of heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase on organic acid production in Aspergillus carbonarius.

    PubMed

    Yang, Lei; Lübeck, Mette; Lübeck, Peter S

    2015-11-01

    Aspergillus carbonarius has a potential as a cell factory for production of various organic acids. In this study, the organic acid profile of A. carbonarius was investigated under different cultivation conditions. Moreover, two heterologous genes, pepck and ppc, which encode phosphoenolpyruvate carboxykinase in Actinobacillus succinogenes and phosphoenolpyruvate carboxylase in Escherichia coli, were inserted individually and in combination in A. carbonarius to enhance the carbon flux toward the reductive TCA branch. Results of transcription analysis and measurement of enzyme activities of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase in the corresponding single and double transformants demonstrated that the two heterologous genes were successfully expressed in A. carbonarius. The production of citric acid increased in all the transformants in both glucose- and xylose-based media at pH higher than 3 but did not increase in the pH non-buffered cultivation compared with the wild type.

  6. Nitrate-Dependent Degradation of Acetone by Alicycliphilus and Paracoccus Strains and Comparison of Acetone Carboxylase Enzymes ▿

    PubMed Central

    Dullius, Carlos Henrique; Chen, Ching-Yuan; Schink, Bernhard

    2011-01-01

    A novel acetone-degrading, nitrate-reducing bacterium, strain KN Bun08, was isolated from an enrichment culture with butanone and nitrate as the sole sources of carbon and energy. The cells were motile short rods, 0.5 to 1 by 1 to 2 μm in size, which gave Gram-positive staining results in the exponential growth phase and Gram-negative staining results in the stationary-growth phase. Based on 16S rRNA gene sequence analysis, the isolate was assigned to the genus Alicycliphilus. Besides butanone and acetone, the strain used numerous fatty acids as substrates. An ATP-dependent acetone-carboxylating enzyme was enriched from cell extracts of this bacterium and of Alicycliphilus denitrificans K601T by two subsequent DEAE Sepharose column procedures. For comparison, acetone carboxylases were enriched from two additional nitrate-reducing bacterial species, Paracoccus denitrificans and P. pantotrophus. The products of the carboxylase reaction were acetoacetate and AMP rather than ADP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cell extracts and of the various enzyme preparations revealed bands corresponding to molecular masses of 85, 78, and 20 kDa, suggesting similarities to the acetone carboxylase enzymes described in detail for the aerobic bacterium Xanthobacter autotrophicus strain Py2 (85.3, 78.3, and 19.6 kDa) and the phototrophic bacterium Rhodobacter capsulatus. Protein bands were excised and compared by mass spectrometry with those of acetone carboxylases of aerobic bacteria. The results document the finding that the nitrate-reducing bacteria studied here use acetone-carboxylating enzymes similar to those of aerobic and phototrophic bacteria. PMID:21841031

  7. Discovery of Small Molecule Isozyme Non-specific Inhibitors of Mammalian Acetyl-CoA Carboxylase 1 and 2

    SciTech Connect

    Corbett, J.; Freeman-Cook, K; Elliott, R; Vajdos, F; Rajamohan, F; Kohls, D; Marr, E; Harwood Jr., H; Esler, W; et al.

    2010-01-01

    Screening Pfizer's compound library resulted in the identification of weak acetyl-CoA carboxylase inhibitors, from which were obtained rACC1 CT-domain co-crystal structures. Utilizing HTS hits and structure-based drug discovery, a more rigid inhibitor was designed and led to the discovery of sub-micromolar, spirochromanone non-specific ACC inhibitors. Low nanomolar, non-specific ACC-isozyme inhibitors that exhibited good rat pharmacokinetics were obtained from this chemotype.

  8. Discovery of small molecule isozyme non-specific inhibitors of mammalian acetyl-CoA carboxylase 1 and 2.

    PubMed

    Corbett, Jeffrey W; Freeman-Cook, Kevin D; Elliott, Richard; Vajdos, Felix; Rajamohan, Francis; Kohls, Darcy; Marr, Eric; Zhang, Hailong; Tong, Liang; Tu, Meihua; Murdande, Sharad; Doran, Shawn D; Houser, Janet A; Song, Wei; Jones, Christopher J; Coffey, Steven B; Buzon, Leanne; Minich, Martha L; Dirico, Kenneth J; Tapley, Susan; McPherson, R Kirk; Sugarman, Eliot; Harwood, H James; Esler, William

    2010-04-01

    Screening Pfizer's compound library resulted in the identification of weak acetyl-CoA carboxylase inhibitors, from which were obtained rACC1 CT-domain co-crystal structures. Utilizing HTS hits and structure-based drug discovery, a more rigid inhibitor was designed and led to the discovery of sub-micromolar, spirochromanone non-specific ACC inhibitors. Low nanomolar, non-specific ACC-isozyme inhibitors that exhibited good rat pharmacokinetics were obtained from this chemotype.

  9. GroE heat shock protein is required for in vivo assembly of recombinant Anabaena ribulose bisphosphate (Ru-P sub 2 ) carboxylase/oxygenase

    SciTech Connect

    Larimer, F.W.; Soper, T.S. )

    1991-03-11

    As a prerequisite for site-directed mutagenesis of a L{sub 8}S{sub 8} form of Ru-P{sub 2} carboxylase, the rbc operon from Anabaena 7120 was placed under control of the tac promoter (tac-rbcLrbcS, bla, ori(pMB1), from pFL260) in E. coli MV1190 (recA). Substantial amounts of insoluble large subunit were produced, but not active enzyme, suggesting that the carboxylase was not being correctly assembled in vivo. Coexpression of rbcLrbcS and the operon encoding the GroESL (HSP10, HSP60) complex from a compatible plasmid (tac-groESgroEL, cat, ori(p15A), from pFL261) resulted in high levels of active, soluble enzyme. Supplementation of rich medium with potassium ions, required for GroE complex function in vitro enhanced recovery of active enzyme. Under optimal expression conditions, active Ru-P{sub 2} carboxylase comprised 7-10% of soluble protein. The recombinant carboxylase, purified to homogeneity, was similar to the enzyme purified from the authentic cyanobacterium. Chaperonins are required for assembly of many complex proteins. The stringent requirement of Anabaena carboxylase for elevated levels of E. coli GroE chaperonin for proper assembly suggests that the GroE complex differs from the Anabaena chaperonin complex that is normally involved in the assembly of this L{sub 8}S{sub 8} carboxylase.

  10. The opportunity for and significance of alteration of ribulose 1,5-bisphosphate carboxylase activities in crop production.

    PubMed

    Hardy, R W; Havelka, U D; Quebedeaux, B

    1978-01-01

    Increased population and the dietary changes accompanying increased affluence are creating a need for a suggested doubling of world cereal grain production (a 3% per year compounding rate) and quadrupling of grain legume production (a 6% per year compounding rate) during this quarter century (1). CO2 enrichment of field-grown crops has demonstrated the possibility of enhancing RuBP carboxylase activity to achieve improved crop production; it increases the production of grain legumes by 50 to 100% and that of cereal grains, for which the studies are less complete, by perhaps 10 to 50%. Results of O2 alteration of growth-room legumes and cereal grains are consistent with the results of CO2 enrichment except for a second role of O2 in assimilate partitioning. It may be necessary to include other components of the system, e.g., additional soil fertility, especially for non-N2-fixing plants, to enable an improved RuBP carboxylase to increase production. No practical method--chemical, genetic, or physical--of improving RuBP carboxylase activity has been reported.

  11. Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase.

    PubMed

    Weaver, K E; Tabita, F R

    1983-11-01

    Several mutants of Rhodopseudomonas sphaeroides defective in the derepression of the enzyme ribulose 1,5-bisphosphate carboxylase have been isolated by using the unstable Tn5 vectors pJB4JI and pRK340. Transpositional insertion mutants obtained with pJB4JI were demonstrated to be incapable of increasing ribulose 1,5-bisphosphate carboxylase/oxygenase levels when grown on butyrate-bicarbonate medium or under conditions of carbon starvation, whereas the wild-type strain increased activity four- to eightfold. When the wild-type strain was starved for carbon in the presence of chloramphenicol, no derepression was observed. Crude extracts from mutant and wild-type strains had distinct and consistent differences in protein content as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic evidence indicated that mutants were defective in the regulation of only one of the two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase synthesized by R. sphaeroides.

  12. Acetyl Coenzyme A Carboxylase Activity in Developing Seedlings and Chloroplasts of Barley and Its Virescens Mutant 1

    PubMed Central

    Thomson, Lawrence W.; Zalik, Saul

    1981-01-01

    Acetyl coenzyme A (CoA) carboxylase activity of whole tissue homogenates and chloroplast preparations was analyzed as the acetyl-CoA-dependent incorporation of [14C]bicarbonate into an acid-stable product. The absolute requirement for ATP and MgCl2, the complete inhibition with avidin, and end-product analysis were consistent with the presence of acetyl-CoA carboxylase activity. Little difference was found between the mutant and normal tissue homogenates from the 1- to 3-day growth stages, during which period both showed a 3-fold increase. However, by 4 days, the activity of the mutant exceeded that of the normal. Fractionation studies showed that the enzyme was a soluble protein present in the stromal fraction of chloroplasts. The biotin content was also highest in the stroma, although it was found in the lamellar fraction as well. For both the mutant and the normal, the highest acetyl-CoA carboxylase activities were obtained in the stromal preparations from 4-day seedlings (54 and 31 nmoles per milligram protein per minute for the mutant and the normal, respectively) with a progressive decline by 6 and 8 days. The difference between the mutant and the normal was not due to the accumulation of an inhibitor in the normal. PMID:16661731

  13. Function of Estrogen Receptor Tryosine Phosphorylation

    DTIC Science & Technology

    1998-07-01

    6219 TITLE: Function of Estrogen Receptor Tryosine Phosphorylation PRINCIPAL INVESTIGATOR: Matthew R. Yudt CONTRACTING ORGANIZATION: University of...Estrogen Receptor Tryosine Phosphorylation ~DAMD17-96-1-6219 6. AUTHOR(S) Matthew R. Yudt 7. PERFORMING ORGANIZATION NAME11S) AND AODRESS(ES...this model, tyrosine 537 (Y537) phosphorylation of one monomer interacts with another tyrosine phosphorylated monomer to constitute an hER dimer

  14. Requirement for Acetyl-CoA Carboxylase in Trypanosoma brucei is Dependent Upon the Growth Environment

    PubMed Central

    Vigueira, Patrick A.; Paul, Kimberly S.

    2013-01-01

    Summary Trypanosoma brucei, the causative agent of human African trypanosomiasis, possesses two fatty acid synthesis pathways: a major de novo synthesis pathway in the ER and a mitochondrial pathway. The 2-carbon donor for both pathways is malonyl-CoA, which is synthesized from acetyl-CoA by Acetyl-CoA Carboxylase (ACC). Here, we show that T. brucei ACC shares the same enzyme architecture and moderate ~30% identity with yeast and human ACCs. ACC is cytoplasmic and appears to be distributed throughout the cell in numerous puncta distinct from glycosomes and other organelles. ACC is active in both bloodstream and procyclic forms. Reduction of ACC activity by RNA interference (RNAi) resulted in a stage-specific phenotype. In procyclic forms, ACC RNAi resulted in 50-75% reduction in fatty acid elongation and a 64% reduction in growth in low lipid media. In bloodstream forms, ACC RNAi resulted in a minor 15% decrease in fatty acid elongation and no growth defect in culture, even in low lipid media. However, ACC RNAi did attenuate virulence in a mouse model of infection. Thus the requirement for ACC in T. brucei is dependent upon the growth environment in two different life cycle stages. PMID:21306439

  15. Evaluation of 3-methylcrotonyl-CoA carboxylase deficiency detected by tandem mass spectrometry newborn screening.

    PubMed

    Koeberl, D D; Millington, D S; Smith, W E; Weavil, S D; Muenzer, J; McCandless, S E; Kishnani, P S; McDonald, M T; Chaing, S; Boney, A; Moore, E; Frazier, D M

    2003-01-01

    Since the addition of tandem mass spectrometry (MS/MS) to the North Carolina Newborn Screening Program, 20 infants with two consecutive elevated 3-hydroxyisovalerylcarnitine (C5OH) levels have been evaluated for evidence of inborn errors of metabolism associated with this metabolite. Ten of these 20 infants had significant concentrations of both 3-hydroxyisovaleric acid and 3-methylcrotonylglycine in their urine, suggestive of 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency. Four of these 10 were infants whose abnormal metabolites were found to be of maternal origin. Of 8 patients with probable 3-MCC deficiency, 7 have been tested and found to have the enzyme deficiency confirmed in lymphoblasts or cultured fibroblasts; one of these 7 infants had only marginally decreased 3-MCC activity in lymphocytes but deficient 3-MCC in fibroblasts. We estimate the incidence of 3-MCC deficiency at 1:64000 live births in North Carolina. We conclude that MS/MS newborn screening will detect additional inborn errors of metabolism, such as 3-MCC deficiency, not traditionally associated with newborn screening. The evaluation of newborns with two abnormally elevated C5OH levels on MS/MS newborn screening should include, at least, urine organic acid analysis by capillary GC-MS and a plasma acylcarnitine profile by MS/MS. Long-term follow-up is needed to determine the outcome of presymptomatically diagnosed patients with 3-MCC deficiency by MS/MS newborn screening.

  16. Pyruvate carboxylase from Rhizobium etli: mutant characterization, nucleotide sequence, and physiological role.

    PubMed Central

    Dunn, M F; Encarnación, S; Araíza, G; Vargas, M C; Dávalos, A; Peralta, H; Mora, Y; Mora, J

    1996-01-01

    Pyruvate carboxylase (PYC), a biotin-dependent enzyme which catalyzes the conversion of pyruvate to oxaloacetate, was hypothesized to play an important anaplerotic role in the growth of Rhizobium etli during serial subcultivation in minimal media containing succinate (S. Encarnación, M. Dunn, K. Willms, and J. Mora, J. Bacteriol. 177:3058-3066, 1995). R. etli and R. tropici pyc::Tn5-mob mutants were selected for their inability to grow in minimal medium with pyruvate as a sole carbon source. During serial subcultivation in minimal medium containing 30 mM succinate, the R. etli parent and pyc mutant strains exhibited similar decreases in growth rate with each subculture. Supplementation of the medium with biotin prevented the growth decrease of the parent but not the mutant strain, indicating that PYC was necessary for the growth of R. etli under these conditions. The R. tropici pyc mutant grew normally in subcultures regardless of biotin supplementation. The symbiotic phenotypes of the pyc mutants from both species were similar to those of the parent strains. The R. etli pyc was cloned, sequenced, and found to encode a 126-kDa protein of 1,154 amino acids. The deduced amino acid sequence is highly homologous to other PYC sequences, and the catalytic domains involved in carboxylation, pyruvate binding, and biotinylation are conserved. The sequence and biochemical data show that the R. etli PYC is a member of the alpha4, homotetrameric, acetyl coenzyme A-activated class of PYCs. PMID:8830693

  17. Activating Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Combination for Improvement of Succinate Production

    PubMed Central

    Tan, Zaigao; Zhu, Xinna; Chen, Jing; Li, Qingyan

    2013-01-01

    Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase (PCK), are responsible for PEP carboxylation. PPC has high substrate affinity and catalytic velocity but wastes the high energy of PEP. PCK has low substrate affinity and catalytic velocity but can conserve the high energy of PEP for ATP formation. In this work, the expression of both the ppc and pck genes was modulated, with multiple regulatory parts of different strengths, in order to investigate the relationship between PPC or PCK activity and succinate production. There was a positive correlation between PCK activity and succinate production. In contrast, there was a positive correlation between PPC activity and succinate production only when PPC activity was within a certain range; excessive PPC activity decreased the rates of both cell growth and succinate formation. These two enzymes were also activated in combination in order to recruit the advantages of each for the improvement of succinate production. It was demonstrated that PPC and PCK had a synergistic effect in improving succinate production. PMID:23747698

  18. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants of RNA turnover. Annual progress report

    SciTech Connect

    Meagher, R.B.

    1993-12-31

    An in vitro degradation system has been developed from petunia and soybean polysomes in order to investigate the mechanisms and determinants controlling RNA turnover in higher plants. This system faithfully degrades soybean ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) mRNA into the same products observed in total RNA preparations. In previous years it was shown that the most stable products represent a nested constellation of fragments, which are shortened from their 3{prime} ends, and have intact 5{prime} ends. Exogenous rbcS RNA tagged with novel 5{prime} sequence 15 or 56 bp long were synthesized in vitro as Sp6 and T7 runoff transcripts, respectively. When added to the system they were degraded faithfully into constellation of products which were 15 or 56 bp longer than the endogenous products, respectively. Detailed kinetics on the appearance of these exogenous products confirmed degradation proceeds in an overall 3{prime} to 5{prime} direction but suggested that there are multiple pathways through which the RNA may be degraded. To further demonstrate a precursor product relationships, in vitro synthesized transcripts truncated at their 3{prime} ends were shown to degrade into the expected smaller fragments previously mapped in the 5{prime} portion of the rbcS RNA.

  19. Slow inactivation of ribulosebisphosphate carboxylase during catalysis is not due to decarbamylation of the catalytic site

    SciTech Connect

    Edmondson, D.L.; Badger, M.R.; Andrews, T.J. )

    1990-08-01

    An investigation was made of the proposal that the slow inactivation of ribulosebisphosphate carboxylase (Rubisco) activity, which occurs during in vitro assays, is due to decarbamylation of the enzyme. The level of carbamylation was compared with catalytic activity during assay conditions in which activity was both increasing and decreasing. A dual isotope procedure was used in which ({sup 3}H)carboxyarabinitol-P{sub 2} measured total active sites and {sup 14}CO{sub 2} reported the level of carbamylation. The efficacy of the procedure was verified both in the presence and in the absence of the substrate D-ribulose-1,5-bisphosphate (ribulose-P{sub 2}). These measurements showed that changes in activity during assays were not correlated with carbamylation status. Inactivation during assays initiated with both fully and partially carbamylated enzyme was not associated with any change in carbamylation level. This implies that the loss of activity during assays is not due to ribulose-P{sub 2} binding and sequestering the E form of the enzyme. Ribulose-P{sub 2} did not appear to alter the equilibrium between carbamylated and uncarbamylated enzyme, but it did slow the rate at which enzyme was both decarbamylated and carbamylated. The most likely explanation for the loss of activity during assays appears to be the sequestration of carbamylated, Mg{sup 2+}-bound active sites by an inhibitor.

  20. Crystalline ribulose bisphosphate carboxylase/oxygenase of high integrity and catalytic activity from Nicotiana tabacum.

    PubMed

    Servaites, J C

    1985-04-01

    Crystalline tobacco (Nicotiana tabacum L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) was prepared using a procedure which protected the enzyme from hydrolysis by endogenous proteases. Leaves were extracted in a buffered medium containing casein, leupeptin, and high concentrations of MgSO4 and NaHCO3. After filtration through ion-exchange resin to remove contaminants, the enzyme was concentrated by precipitation with polyethylene glycol and crystal formation was induced by low-salt dialysis. The crystalline enzyme had a measured specific activity of 1.7 mumol CO2 mg protein-1 min-1, and about 93% of the enzyme could be activated with Mg2+ and CO2. Crystalline enzyme prepared in the absence of casein exhibited an activity which was only one-third of this rate and only about 70% of the enzyme could be activated with Mg2+ and CO2. Casein-extracted enzyme was resolved into distinct bands corresponding to the large (55,000) and small (14,000) subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit of enzyme prepared according to the latter procedure was found to be composed of five different polypeptides of slightly decreasing molecular weight. Only about one-third of the large subunits were of the 55,000 molecular weight type. No differences between the two preparations were observed in the Km (CO2) and apparent Km (ribulose bisphosphate).

  1. [Efficacy of thiamine pyrophosphate or carboxylase in the salvage of diabetic foot].

    PubMed

    Carmona-Cervantes, J

    2014-01-01

    Diabetic foot represents one of the most common complications in patients with a long standing disease. The etiology is neuropathy, infections and ischemia that together contribute to the sequence of tissue necrosis, ulceration and gangrene. Since treatment is very difficult, we must look for several options to solve these problems caused by chronic hyperglycemia. Thiamine pyrophosphate or carboxylase perform multiple metabolic and non-metabolic activities that are considered important in the resolution of diabetic impairments, therefore, this work shows the results when using it in patients with diabetic foot. 29 patients with diabetic foot were treated between January 1998 and July 2012: 19 Wagner type III and 12 Wagner type IV. Management was the administration of antibiotics, partial surgical procedures and thiamine pyrophosphate. The infectious process was controlled, the appearance of granulation tissue and scarring of the lesion in a period of 2 to 6 months depending on the severity of the problem. Given the clinical data and evolution of the patients, we conclude that the administration of thiamine pyrophosphate was able to control metabolic and non-metabolic dysfunctions that lead to complications in diabetic patients, therefore we must consider it a tool in the treatment of diabetic patients in general and for diabetic foot salvage in particular.

  2. Inhibition of acetyl-coenzyme A carboxylase by two classes of grass-selective herbicides

    SciTech Connect

    Rendina, A.R.; Craig-Kennard, A.C.; Beaudoin, J.D.; Breen, M.K. )

    1990-05-01

    The selective grass herbicides diclofop, haloxyfop, and trifop (((aryloxy)phenoxy)propionic acids) and alloxydim, sethoxydim, and clethodim (cyclohexanediones) are potent, reversible inhibitors of acetyl-coenzyme A carboxylase (ACC) partially purified from barley, corn, and wheat. Although inhibition of the wheat enzyme by clethodim and diclofop is noncompetitive versus each of the substrates adenosine triphosphate (ATP), HCO{sub 3}{sup {minus}}, and acetyl-coenzyme A (acetyl-CoA), diclofop and clethodim are nearly competitive versus acetyl-CoA since the level of inhibition is most sensitive to the concentration of acetyl-CoA (K{sub is} < K{sub ii}). To conclusively show whether the herbicides interact at the biotin carboxylation site or the carboxyl transfer site, the inhibition of isotope exchange and partial reactions catalyzed at each site was studied with the wheat enzyme. Only the ({sup 14}C)acetyl-CoA-malonyl-CoA exchange and decarboxylation of ({sup 14}C)malonyl-CoA reactions are strongly inhibited by clethodim and diclofop, suggesting that the herbicides interfere with the carboxyl transfer site rather than the biotin carboxylation site of the enzyme. Double-inhibition studies with diclofop and clethodim suggest that the ((aryloxy)phenoxy)propionic acid and cyclohexanedione herbicides may bind to the same region of the enzyme.

  3. Acetyl-CoA carboxylase alpha is essential to breast cancer cell survival.

    PubMed

    Chajès, Véronique; Cambot, Marie; Moreau, Karen; Lenoir, Gilbert M; Joulin, Virginie

    2006-05-15

    Activation of de novo fatty acid synthesis is a characteristic feature of cancer cells. We have recently described an interaction between acetyl-CoA carboxylase alpha (ACCalpha), a key enzyme in fatty acid synthesis, and BRCA1, which indicates a possible connection between lipid synthesis and genetic factors involved in susceptibility to breast and ovarian cancers. For this reason, we explored the role of ACCalpha in breast cancer cell survival using an RNA interference (RNAi) approach. We show that specific silencing of either the ACCalpha or the fatty acid synthase (FAS) genes in cancer cells results in a major decrease in palmitic acid synthesis. Depletion of the cellular pool of palmitic acid is associated with induction of apoptosis concomitant with the formation of reactive oxygen species (ROS) and mitochondrial impairment. Expression of a small interfering RNA (siRNA)-resistant form of ACCalpha mRNA prevented the effect of ACCalpha-RNAi but failed to prevent the effect of FAS gene silencing. Furthermore, supplementation of the culture medium with palmitate or with the antioxidant vitamin E resulted in the complete rescue of cells from both ACCalpha and FAS siRNA-induced apoptosis. Finally, human mammary epithelial cells are resistant to RNAi against either ACCalpha or FAS. These data confirm the importance of lipogenesis in cancer cell survival and indicate that this pathway represents a key target for antineoplastic therapy that, however, might require specific dietary recommendation for full efficacy.

  4. 3-methylcrotonyl-CoA carboxylase deficiency: Clinical, biochemical, enzymatic and molecular studies in 88 individuals

    PubMed Central

    2012-01-01

    Background Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine metabolism caused by mutations in MCCC1 or MCCC2 encoding the α and β subunit of MCC, respectively. The phenotype is highly variable ranging from acute neonatal onset with fatal outcome to asymptomatic adults. Methods We report clinical, biochemical, enzymatic and mutation data of 88 MCC deficient individuals, 53 identified by newborn screening, 26 diagnosed due to clinical symptoms or positive family history and 9 mothers, identified following the positive newborn screening result of their baby. Results Fifty-seven percent of patients were asymptomatic while 43% showed clinical symptoms, many of which were probably not related to MCC deficiency but due to ascertainment bias. However, 12 patients (5 of 53 identified by newborn screening) presented with acute metabolic decompensations. We identified 15 novel MCCC1 and 16 novel MCCC2 mutant alleles. Additionally, we report expression studies on 3 MCCC1 and 8 MCCC2 mutations and show an overview of all 132 MCCC1 and MCCC2 variants known to date. Conclusions Our data confirm that MCC deficiency, despite low penetrance, may lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting the clinical course. PMID:22642865

  5. Improvement of the phosphoenolpyruvate carboxylase activity of Phaeodactylum tricornutum PEPCase 1 through protein engineering.

    PubMed

    Chang, Kwang Suk; Jeon, Hancheol; Seo, Seungbeom; Lee, Yew; Jin, EonSeon

    2014-06-10

    In order to mitigate CO2 accumulation and decrease the rate of global warming and climate change, we previously presented a strategy for the development of an efficient CO2 capture and utilization system. The system employs two recombinant enzymes, carbonic anhydrase and phosphoenolpyruvate carboxylase, which were originated from microalgae. Although utilization of this integrated system would require a large quantity of high quality PEPCase protein, such quantities could be produced by increasing the solubility of the Phaeodactylum tricornutum PEPCase 1 (PtPEPCase 1) protein in the Escherichia coli heterologous expression system. We first expressed the putative mitochondria targeting peptide- and chloroplast transit peptide-truncated proteins of PtPEPCase 1, mPtPEPCase 1 and cPtPEPCase 1, respectively, in E. coli. After affinity chromatography, the amount of purified PEPCase protein from 500mL of E. coli culture was greatest for cPtPEPCase 1 (1.99mg), followed by mPtPEPCase 1 (0.82mg) and PtPEPCase 1 (0.61mg). Furthermore, the enzymatic activity of mPtPEPCase 1 and cPtPEPCase 1 showed approximately 1.6-fold (32.19 units/mg) and 3-fold (59.48 units/mg) increases, respectively. Therefore, cPtPEPCase 1 purified using the E. coli heterogeneous expression system could be a strong candidate for a platform technology to capture CO2 and produce value-added four-carbon platform chemicals.

  6. Characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in Corynebacterium glutamicum.

    PubMed

    Nagano-Shoji, Megumi; Hamamoto, Yuma; Mizuno, Yuta; Yamada, Ayuka; Kikuchi, Masaki; Shirouzu, Mikako; Umehara, Takashi; Yoshida, Minoru; Nishiyama, Makoto; Kosono, Saori

    2017-03-03

    Protein Nε-acylation is emerging as a ubiquitous post-translational modification. In Corynebacterium glutamicum, which is utilized for industrial production of L-glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. Here, we characterized the acylation of phosphoenolpyruvate carboxylase (PEPC), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction. We showed that acetylation of PEPC at lysine 653 decreased enzymatic activity, leading to reduced glutamate production. An acetylation-mimic (KQ) mutant of K653 showed severely reduced glutamate production, while the corresponding KR mutant showed normal production levels. Using an acetyllysine-incorporated PEPC protein, we verified that K653-acetylation negatively regulates PEPC activity. In addition, NCgl0616, a sirtuin-type deacetylase, deacetylated K653-acetylated PEPC in vitro. Interestingly, the specific activity of PEPC was increased during glutamate overproduction, which was blocked by the K653R mutation or deletion of sirtuin-type deacetylase homologues. These findings suggested that deacetylation of K653 by NCgl0616 likely plays a role in the activation of PEPC, which maintains carbon flux under glutamate-producing conditions. PEPC deletion increased protein acetylation levels in cells under glutamate-producing conditions, supporting our hypothesis that PEPC is responsible for a large carbon flux change under glutamate-producing conditions. This article is protected by copyright. All rights reserved.

  7. Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti Rm1021.

    PubMed

    Dunn, M F; Araíza, G; Finan, T M

    2001-11-01

    The gene encoding pyruvate carboxylase (pyc) was isolated from a Sinorhizobium meliloti Rm1021 cosmid bank by complementation of a Rhizobium tropici pyc mutant. PYC-negative mutants of S. meliloti Rm1021 were isolated by transposon mutagenesis and were unable to grow with glucose or pyruvate as sole carbon sources, but were symbiotically competent in combination with alfalfa plants. PYC activity assays, pyc::lacZ gene fusion studies and an in vivo biotinylation assay showed that PYC activity in S. meliloti was dependent mainly on biotin availability and not on changes in gene transcription. The subunit and holo-enzyme molecular masses of the S. meliloti PYC indicated that the enzyme was an alpha4 homotetramer. The S. meliloti PYC had a high apparent Ka (0.23 mM) for the allosteric activator acetyl-CoA and was product-inhibited by sub-millimolar concentrations of oxaloacetate. In contrast to other bacterial alpha4-PYCs which have been characterized, the S. meliloti enzyme was not strongly inhibited by L-aspartate.

  8. Allosteric Inhibition of Phosphoenolpyruvate Carboxylases is Determined by a Single Amino Acid Residue in Cyanobacteria

    PubMed Central

    Takeya, Masahiro; Hirai, Masami Yokota; Osanai, Takashi

    2017-01-01

    Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme for CO2 fixation and primary metabolism in photosynthetic organisms including cyanobacteria. The kinetics and allosteric regulation of PEPCs have been studied in many organisms, but the biochemical properties of PEPC in the unicellular, non-nitrogen-fixing cyanobacterium Synechocystis sp. PCC 6803 have not been clarified. In this study, biochemical analysis revealed that the optimum pH and temperature of Synechocystis 6803 PEPC proteins were 7.3 and 30 °C, respectively. Synechocystis 6803 PEPC was found to be tolerant to allosteric inhibition by several metabolic effectors such as malate, aspartate, and fumarate compared with other cyanobacterial PEPCs. Comparative sequence and biochemical analysis showed that substitution of the glutamate residue at position 954 with lysine altered the enzyme so that it was inhibited by malate, aspartate, and fumarate. PEPC of the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 was purified, and its activity was inhibited in the presence of malate. Substitution of the lysine at position 946 (equivalent to position 954 in Synechocystis 6803) with glutamate made Anabaena 7120 PEPC tolerant to malate. These results demonstrate that the allosteric regulation of PEPC in cyanobacteria is determined by a single amino acid residue, a characteristic that is conserved in different orders. PMID:28117365

  9. Tyrosine phosphorylation of WW proteins

    PubMed Central

    Reuven, Nina; Shanzer, Matan

    2015-01-01

    A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein–protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins. PMID:25627656

  10. Phosphorylation of tau is regulated by PKN.

    PubMed

    Taniguchi, T; Kawamata, T; Mukai, H; Hasegawa, H; Isagawa, T; Yasuda, M; Hashimoto, T; Terashima, A; Nakai, M; Mori, H; Ono, Y; Tanaka, C

    2001-03-30

    For the phosphorylation state of microtubule-associated protein, tau plays a pivotal role in regulating microtubule networks in neurons. Tau promotes the assembly and stabilization of microtubules. The potential for tau to bind to microtubules is down-regulated after local phosphorylation. When we investigated the effects of PKN activation on tau phosphorylation, we found that PKN triggers disruption of the microtubule array both in vitro and in vivo and predominantly phosphorylates tau in microtubule binding domains (MBDs). PKN has a catalytic domain highly homologous to protein kinase C (PKC), a kinase that phosphorylates Ser-313 (= Ser-324, the number used in this study) in MBDs. Thus, we identified the phosphorylation sites of PKN and PKC subtypes (PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -zeta, and -lambda) in MBDs. PKN phosphorylates Ser-258, Ser-320, and Ser-352, although all PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352. There is a PKN-specific phosphorylation site, Ser-320, in MBDs. HIA3, a novel phosphorylation-dependent antibody recognizing phosphorylated tau at Ser-320, showed immunoreactivity in Chinese hamster ovary cells expressing tau and the active form of PKN, but not in Chinese hamster ovary cells expressing tau and the inactive form of PKN. The immunoreactivity for phosphorylated tau at Ser-320 increased in the presence of a phosphatase inhibitor, FK506 treatment, which means that calcineurin (protein phosphatase 2B) may be involved in dephosphorylating tau at Ser-320 site. We also noted that PKN reduces the phosphorylation recognized by the phosphorylation-dependent antibodies AT8, AT180, and AT270 in vivo. Thus PKN serves as a regulator of microtubules by specific phosphorylation of tau, which leads to disruption of tubulin assembly.

  11. Epigenetic regulation of pyruvate carboxylase gene expression in the postpartum liver.

    PubMed

    Walker, C G; Crookenden, M A; Henty, K M; Handley, R R; Kuhn-Sherlock, B; White, H M; Donkin, S S; Snell, R G; Meier, S; Heiser, A; Loor, J J; Mitchell, M D; Roche, J R

    2016-07-01

    Hepatic gluconeogenesis is essential for maintenance of whole body glucose homeostasis and glucose supply for mammary lactose synthesis in the dairy cow. Upregulation of the gluconeogenic enzyme pyruvate carboxylase (PC) during the transition period is vital in the adaptation to the greater glucose demands associated with peripartum lactogenesis. The objective of this study was to determine if PC transcription in hepatocytes is regulated by DNA methylation and if treatment with a nonsteroidal anti-inflammatory drug (NSAID) alters methylation of an upstream DNA sequence defined as promoter 1. Dairy cows were left untreated (n=20), or treated with a NSAID during the first 5 d postcalving (n=20). Liver was biopsied at d 7 precalving and d 7, 14, and 28 postcalving. Total PC and transcript specific gene expression was quantified using quantitative PCR and DNA methylation of promoter 1 was quantified using bisulfite Sanger sequencing. Expression of PC changed over the transition period, with increased expression postcalving occurring concurrently with increased circulating concentration of nonesterified fatty acids. The DNA methylation percentage was variable at all sites quantified and ranged from 21 to 54% across the 15 CpG dinucleotides within promoter 1. The DNA methylation at wk 1 postcalving, however, was not correlated with gene expression of promoter 1-regulated transcripts and we did not detect an effect of NSAID treatment on DNA methylation or PC gene expression. Our results do not support a role for DNA methylation in regulating promoter 1-driven gene expression of PC at wk 1 postcalving. Further research is required to determine the mechanisms regulating increased PC expression over the transition period.

  12. Unusual ribulose 1,5-bisphosphate carboxylase/oxygenase of anoxic Archaea.

    PubMed

    Watson, G M; Yu, J P; Tabita, F R

    1999-03-01

    The predominant pool of organic matter on earth is derived from the biological reduction and assimilation of carbon dioxide gas, catalyzed primarily by the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). By virtue of its capacity to use molecular oxygen as an alternative and competing gaseous substrate, the catalytic efficiency of RubisCO and the enzyme's ability to assimilate CO2 may be severely limited, with consequent environmental and agricultural effects. Recent genomic sequencing projects, however, have identified putative RubisCO genes from anoxic Archaea. In the present study, these potential RubisCO sequences, from Methanococcus jannaschii and Archaeoglobus fulgidus, were analyzed in order to ascertain whether such sequences might encode functional proteins. We also report the isolation and properties of recombinant RubisCO using sequences obtained from the obligately anaerobic hyperthermophilic methanogen M. jannaschii. This is the first description of an archaeal RubisCO sequence; this study also represents the initial characterization of a RubisCO molecule that has evolved in the absence of molecular oxygen. The enzyme was shown to be a homodimer whose deduced sequence, along with other recently obtained archaeal RubisCO sequences, differs substantially from those of known RubisCO molecules. The recombinant M. jannaschii enzyme has a somewhat low, but reasonable kcat, however, unlike previously isolated RubisCO molecules, this enzyme is very oxygen sensitive yet it is stable to hyperthermal temperatures and catalyzes the formation of the expected carboxylation product. Despite inhibition by oxygen, this unusual RubisCO still catalyzes a weak yet demonstrable oxygenase activity, with perhaps the lowest capacity for CO2/O2 discrimination ever encountered for any RubisCO.

  13. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    PubMed

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum.

  14. Cloning of human acetyl-CoA carboxylase-beta and its unique features.

    PubMed Central

    Ha, J; Lee, J K; Kim, K S; Witters, L A; Kim, K H

    1996-01-01

    Acetyl-CoA carboxylase, which has a molecular mass of 265 kDa (ACC-alpha), catalyzes the rate-limiting step in the biosynthesis of long-chain fatty acids. In this study we report the complete amino acid sequence and unique features of an isoform of ACC with a molecular mass of 275 kDa (ACC-beta), which is primarily expressed in heart and skeletal muscles. In these tissues, ACC-beta may be involved in the regulation of fatty acid oxidation, rather than fatty acid biosynthesis. ACC-beta contains an amino acid sequence at the N terminus which is about 200 amino acids long and may be uniquely related to the role of ACC-beta in controlling carnitine palmitoyltransferase I activity and fatty acid oxidation by mitochondria. If we exclude this unique sequence at the N terminus the two forms of ACC show about 75% amino acid identity. All of the known functional domains of ACC are found in the homologous regions. Human ACC-beta cDNA has an open reading frame of 7,343 bases, encoding a protein of 2,458 amino acids, with a calculated molecular mass of 276,638 Da. The mRNA size of human ACC-beta is approximately 10 kb and is primarily expressed in heart and skeletal muscle tissues, whereas ACC-alpha mRNA is detected in all tissues tested. A fragment of ACC-beta cDNA was expressed in Escherichia coli and antibodies against the peptide were generated to establish that the cDNA sequence that we cloned is that for ACC-beta. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8876158

  15. Synthesis of catalytically active form III ribulose 1,5-bisphosphate carboxylase/oxygenase in archaea.

    PubMed

    Finn, Michael W; Tabita, F Robert

    2003-05-01

    Ribulose 1,5 bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the biological reduction and assimilation of carbon dioxide gas to organic carbon; it is the key enzyme responsible for the bulk of organic matter found on earth. Until recently it was believed that there are only two forms of RubisCO, form I and form II. However, the recent completion of several genome-sequencing projects uncovered open reading frames resembling RubisCO in the third domain of life, the archaea. Previous work and homology comparisons suggest that these enzymes represent a third form of RubisCO, form III. While earlier work indicated that two structurally distinct recombinant archaeal RubisCO proteins catalyzed bona fide RubisCO reactions, it was not established that the rbcL genes of anaerobic archaea can be transcribed and translated to an active enzyme in the native organisms. In this report, it is shown not only that Methanococcus jannaschii, Archaeoglobus fulgidus, Methanosarcina acetivorans, and Methanosarcina barkeri possess open reading frames with the residues required for catalysis but also that the RubisCO protein from these archaea accumulates in an active form under normal growth conditions. In addition, the form III RubisCO gene (rbcL) from M. acetivorans was shown to complement RubisCO deletion strains of Rhodobacter capsulatus and Rhodobacter sphaeroides under both photoheterotrophic and photoautotrophic growth conditions. These studies thus indicate for the first time that archaeal form III RubisCO functions in a physiologically significant fashion to fix CO(2). Furthermore, recombinant M. jannaschii, M. acetivorans, and A. fulgidus RubisCO possess unique properties with respect to quaternary structure, temperature optima, and activity in the presence of molecular oxygen compared to the previously described Thermococcus kodakaraensis and halophile proteins.

  16. Glucose and fat metabolism in adipose tissue of acetyl-CoA carboxylase 2 knockout mice

    PubMed Central

    Oh, WonKeun; Abu-Elheiga, Lutfi; Kordari, Parichher; Gu, Zeiwei; Shaikenov, Tattym; Chirala, Subrahmanyam S.; Wakil, Salih J.

    2005-01-01

    Acc2-/- mutant mice, when fed a high-fat/high-carbohydrate (HF/HC) diet, were protected against diet-induced obesity and diabetes. To investigate the role of acetyl-CoA carboxylase 2 (ACC2) in the regulation of energy metabolism in adipose tissues, we studied fatty acid and glucose oxidation in primary cultures of adipocytes isolated from wild-type and Acc2-/- mutant mice fed either normal chow or a HF/HC diet. When fed normal chow, oxidation of [14C]palmitate in adipocytes of Acc2-/- mutant mice was ≈80% higher than in adipocytes of WT mice, and it remained significantly higher in the presence of insulin. Interestingly, in addition to increased fatty acid oxidation, we also observed increased glucose oxidation in adipocytes of Acc2-/- mutant mice compared with that of WT mice. When fed a HF/HC diet for 4-5 months, adipocytes of Acc2-/- mutant mice maintained a 25% higher palmitate oxidation and a 2-fold higher glucose oxidation than WT mice. The mRNA level of glucose transporter 4 (GLUT4) decreased several fold in the adipose tissue of WT mice fed a HF/HC diet; however, in the adipose tissue of Acc2-/- mutant mice, it was 7-fold higher. Moreover, lipolysis activity was higher in adipocytes of Acc2-/- mutant mice compared with that in WT mice. These findings suggest that continuous fatty acid oxidation in the adipocytes of Acc2-/- mutant mice, combined with a higher level of glucose oxidation and a higher rate of lipolysis, are major factors leading to efficient maintenance of insulin sensitivity and leaner Acc2-/- mutant mice. PMID:15677334

  17. Multiple Inter-Kingdom Horizontal Gene Transfers in the Evolution of the Phosphoenolpyruvate Carboxylase Gene Family

    PubMed Central

    Wang, Wen; Su, Bing

    2012-01-01

    Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought. PMID:23251445

  18. Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

    PubMed Central

    Galka, Marek M.; Rajagopalan, Nandhakishore; Buhrow, Leann M.; Nelson, Ken M.; Switala, Jacek; Cutler, Adrian J.; Palmer, David R. J.; Loewen, Peter C.; Abrams, Suzanne R.; Loewen, Michele C.

    2015-01-01

    Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation. PMID:26197050

  19. Towards efficient photosynthesis: overexpression of Zea mays phosphoenolpyruvate carboxylase in Arabidopsis thaliana.

    PubMed

    Kandoi, Deepika; Mohanty, Sasmita; Govindjee; Tripathy, Baishnab C

    2016-12-01

    Plants with C4 photosynthesis are efficient in carbon assimilation and have an advantage over C3 photosynthesis. In C4 photosynthesis, the primary CO2 fixation is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Here, we show that overexpression of Zea mays PEPC cDNA, under the control of (35)S promoter, in Arabidopsis thaliana resulted in ~7-10 fold higher protein abundance and ~7-10 fold increase in PEPC activity in the transgenic lines than that in the vector control. We suggest that overexpression of PEPC played an anaplerotic role to increase the supply of 4-carbon carboxylic acids, which provided carbon skeletons for increased amino acid and protein synthesis. Higher protein content must have been responsible for increased metabolic processes including chlorophyll biosynthesis, photosynthesis, and respiration. Consequently, the PEPC-overexpressed transgenic plants had higher chlorophyll content, enhanced electron transport rate (ETR), lower non-photochemical quenching (NPQ) of chlorophyll a fluorescence, and a higher performance index (PI) than the vector control. Consistent with these observations, the rate of CO2 assimilation, the starch content, and the dry weight of PEPC-overexpressed plants increased by 14-18 %, 10-18 %, and 6.5-16 %, respectively. Significantly, transgenics were tolerant to salt stress as they had increased ability to synthesize amino acids, including the osmolyte proline. NaCl (150 mM)-treated transgenic plants had higher variable to maximum Chl a fluorescence (F v/F m) ratio, higher PI, higher ETR, and lower NPQ than the salt-treated vector controls. These results suggest that expression of C4 photosynthesis enzyme(s) in a C3 plant can improve its photosynthetic capacity with enhanced tolerance to salinity stress.

  20. Molecular Evolution of the Small Subunit of Ribulose Bisphosphate Carboxylase: Nucleotide Substitution and Gene Conversion

    PubMed Central

    Meagher, R. B.; Berry-Lowe, S.; Rice, K.

    1989-01-01

    The nucleotide sequences encoding the mature portion of 31 ribulose 1,5-bisphosphate carboxylase small subunit (SSU) genes from 17 genera of plants, green algae and cyanobacteria were examined. Among the 465 pairwise sequence comparisons, SSU multigene family members within the same species were more similar to each other in nonsynonymous or replacement nucleotide substitutions (RNS) than they were to SSU sequences in any other organism. The concerted evolution of independent SSU gene lineages within closely related plant species suggests that homogenization of RNS positions has occurred at least once in the life of each genus. The rate of expected RNS among mature SSU sequences was calculated to be 1.25 X 10(-9)/site/yr for the first 70 million years (MY) of divergence with a significant slowing to 0.13 X 10(-9)/site/yr for the next 1,400 MY. The data suggest that mature SSU sequences do not accumulate more than 20% differences in the RNS positions without compensatory changes in other components of this enzyme system. During the first 70 MY of divergence between species, the rate of expected synonymous or silent nucleotide substitutions (SNS) is ~6.6 X 10(-9)/site/yr. This is five times the RNS rate and is similar to the silent rate observed in animals. In striking contrast, SNS and RNS do not show this correlation among SSU gene family members within a species. A mechanism involving gene conversion within the exons followed by selection for biased gene conversion products with conservation of RNS positions and divergence of SNS positions is discussed. A SSU gene tree based on corrected RNS for 31 SSU sequences is presented and agrees well with a species tree based on morphological and cytogenetic traits for the 17 genera examined. SSU gene comparisons may be useful in predicting phylogenetic relationships and in some cases divergence times of various plant, algal and cyanobacterial species. PMID:2515110

  1. Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

    DOEpatents

    Houtz, Robert L.

    1999-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

  2. Starch phosphorylation: insights and perspectives.

    PubMed

    Mahlow, Sebastian; Orzechowski, Sławomir; Fettke, Joerg

    2016-07-01

    During starch metabolism, the phosphorylation of glucosyl residues of starch, to be more precise of amylopectin, is a repeatedly observed process. This phosphorylation is mediated by dikinases, the glucan, water dikinase (GWD) and the phosphoglucan, water dikinase (PWD). The starch-related dikinases utilize ATP as dual phosphate donor transferring the terminal γ-phosphate group to water and the β-phosphate group selectively to either C6 position or C3 position of a glucosyl residue within amylopectin. By the collaborative action of both enzymes, the initiation of a transition of α-glucans from highly ordered, water-insoluble state to a less order state is realized and thus the initial process of starch degradation. Consequently, mutants lacking either GWD or PWD reveal a starch excess phenotype as well as growth retardation. In this review, we focus on the increased knowledge collected over the last years related to enzymatic properties, the precise definition of the substrates, the physiological implications, and discuss ongoing questions.

  3. Leucine 332 influences the CO2/O2 specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.

    PubMed Central

    Lee, G. J.; McDonald, K. A.; McFadden, B. A.

    1993-01-01

    The role of Leu 332 in ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans was investigated by site-directed mutagenesis. Substitutions of this residue with Met, Ile, Val, Thr, or Ala decreased the CO2/O2 specificity factor by as much as 67% and 96% for the Ile mutant in the presence of Mg2+ and Mn2+, respectively. For the Met, Ile, and Ala mutants in the presence of Mg2+, no loss of oxygenase activity was observed despite the loss of greater than 65% of the carboxylase activity relative to the wild-type enzyme. In the presence of Mn2+, carboxylase activities for mutant enzymes were reduced to approximately the same degree as was observed in the presence of Mg2+, although oxygenase activities were also reduced to similar extents as carboxylase activities. Only minor changes in Km(RuBP) were observed for all mutants in the presence of Mg2+ relative to the wild-type enzyme, indicating that Leu 332 does not function in RuBP binding. These results suggest that in the presence of Mg2+, Leu 332 contributes to the stabilization of the transition state for the carboxylase reaction, and demonstrate that it is possible to affect only one of the activities of this bifunctional enzyme. PMID:8358297

  4. MEDICA 16 inhibits hepatic acetyl-CoA carboxylase and reduces plasma triacylglycerol levels in insulin-resistant JCR: LA-cp rats.

    PubMed

    Atkinson, Laura L; Kelly, Sandra E; Russell, James C; Bar-Tana, Jacob; Lopaschuk, Gary D

    2002-05-01

    Intracellular triacylglycerol (TG) content of liver and skeletal muscle contributes to insulin resistance, and a significant correlation exists between TG content and the development of insulin resistance. Because acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme for liver fatty acid biosynthesis and a key regulator of muscle fatty acid oxidation, we examined whether ACC plays a role in the accumulation of intracellular TG. We also determined the potential role of 5'-AMP-activated protein kinase (AMPK) in this process, since it can phosphorylate and inhibit ACC activity in both liver and muscle. TG content, ACC, and AMPK were examined in the liver and skeletal muscle of insulin-resistant JCR:LA-cp rats during the time frame when insulin resistance develops. At 12 weeks of age, there was a threefold elevation in liver TG content and a sevenfold elevation in skeletal muscle TG content. Hepatic ACC activity was significantly elevated in 12-week-old JCR:LA-cp rats compared with lean age-matched controls (8.75 +/- 0.53 vs. 3.30 +/- 0.18 nmol. min(-1). mg(-1), respectively), even though AMPK activity was also increased. The observed increase in hepatic ACC activity was accompanied by a 300% increase in ACC protein expression. There were no significant differences in ACC activity, ACC protein expression, or AMPK activity in the skeletal muscle of the 12-week JCR:LA-cp rats. Treatment of 12-week JCR:LA-cp rats with MEDICA 16 (an ATP-citrate lyase inhibitor) resulted in a decrease in hepatic ACC and AMPK activities, but had no effect on skeletal muscle ACC and AMPK. Our data suggest that alterations in ACC or AMPK activity in muscle do not contribute to the development of insulin resistance. However, increased liver ACC activity in the JCR:LA-cp rat appears to contribute to the development of lipid abnormalities, although this increase does not appear to occur secondary to a decrease in AMPK activity.

  5. Unequal synthesis and differential degradation of propionyl CoA carboxylase subunits in cells from normal and propionic acidemia patients.

    PubMed Central

    Ohura, T; Kraus, J P; Rosenberg, L E

    1989-01-01

    We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme's two protein subunits (alpha and beta) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase's alpha and beta subunits. Analysis of pccA lines revealed the absence of alpha chain mRNA in three and an abnormally small alpha-mRNA in a fourth. Despite the presence of normal beta-mRNA in each of these pccA lines, there was complete absence of both alpha and beta protein subunits under steady state conditions, even though new synthesis and mitochondrial import of beta precursors was normal. Results in nine pccBC lines revealed normal alpha mRNA in each, while the amounts of beta-mRNA were distinctly reduced in every case. Correspondingly, alpha protein subunits were present in normal amounts at steady-state, but beta subunits were uniformly decreased. In addition, in six of the nine beta deficient cell lines, partially degraded beta-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas alpha chains were stable over the four hour interval studied, beta chains--initially synthesized in large excess over alpha chains--were degraded rapidly reaching equivalence with alpha chains after two hours.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2741949

  6. Immobilization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase: a step toward carbon dioxide fixation bioprocess.

    PubMed

    Chakrabarti, Subhra; Bhattacharya, Sumana; Bhattacharya, Sanjoy K

    2003-03-20

    Immobilization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from spinach leaves is described. This enzyme enables the fixation of carbon dioxide on a five-carbon sugar D-ribulose-1,5-bisphosphate (RuBP). Two different immobilization methods were employed: dicyclohexylcarbodiimide coupling on nylon membrane matrix and dimethylpimelimidate immobilization on protein A agarose. The reusability of immobilized enzymes, coupling efficiency, and temperature-activity relationship of soluble and immobilized Rubisco are presented. The immobilization imparted greater thermal and storage stability. The thermal deactivation rates of the immobilized enzymes were considerably lower than those of the soluble enzyme.

  7. Suppression of 5'-nucleotidase enzymes promotes AMP-activated protein kinase (AMPK) phosphorylation and metabolism in human and mouse skeletal muscle.

    PubMed

    Kulkarni, Sameer S; Karlsson, Håkan K R; Szekeres, Ferenc; Chibalin, Alexander V; Krook, Anna; Zierath, Juleen R

    2011-10-07

    The 5'-nucleotidase (NT5) family of enzyme dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. We hypothesized that gene silencing of NT5 enzymes to increase the intracellular availability of AMP would increase AMP-activated protein kinase (AMPK) activity and metabolism. We determined the role of cytosolic NT5 in metabolic responses linked to the development of insulin resistance in obesity and type 2 diabetes. Using siRNA to silence NT5C2 expression in cultured human myotubes, we observed a 2-fold increase in the AMP/ATP ratio, a 2.4-fold increase in AMPK phosphorylation (Thr(172)), and a 2.8-fold increase in acetyl-CoA carboxylase phosphorylation (Ser(79)) (p < 0.05). siRNA silencing of NT5C2 expression increased palmitate oxidation by 2-fold in the absence and by 8-fold in the presence of 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside. This was paralleled by an increase in glucose transport and a decrease in glucose oxidation, incorporation into glycogen, and lactate release from NT5C2-depleted myotubes. Gene silencing of NT5C1A by shRNA injection and electroporation in mouse tibialis anterior muscle reduced protein content (60%; p < 0.05) and increased phosphorylation of AMPK (60%; p < 0.05) and acetyl-CoA carboxylase (50%; p < 0.05) and glucose uptake (20%; p < 0.05). Endogenous expression of NT5C enzymes inhibited basal lipid oxidation and glucose transport in skeletal muscle. Reduction of 5'-nucleotidase expression or activity may promote metabolic flexibility in type 2 diabetes.

  8. FT-IR analysis of phosphorylated protein

    NASA Astrophysics Data System (ADS)

    Ishii, Katsunori; Yoshihashi, Sachiko S.; Chihara, Kunihiro; Awazu, Kunio

    2004-09-01

    Phosphorylation and dephosphorylation, which are the most remarkable posttranslational modifications, are considered to be important chemical reactions that control the activation of proteins. We examine the phosphorylation analysis method by measuring the infrared absorption peak of phosphate group that observed at about 1070cm-1 (9.4μm) with Fourier Transform Infrared Spectrometer (FT-IR). This study indicates that it is possible to identify a phosphorylation by measuring the infrared absorption peak of phosphate group observed at about 1070 cm-1 with FT-IR method. As long as target peptides have the same amino acid sequence, it is possible to identify the phosphorylated sites (threonine, serine and tyrosine).

  9. Inactivation of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and spinach with the new affinity label 2-bromo-1,5-dihydroxy-3-pentanone 1,5-bisphosphate

    SciTech Connect

    Donnelly, M.I.; Hartman, F.C.

    1981-11-16

    In an attempt to identify the active-site base believed to initiate catalysis by ribulosebisphosphate carboxylase, we have synthesized 2-bromo-1, 5-dihydroxy-3-pentanone 1,5-bisphosphate, a reactive analogue of a postulated intermediate of carboxylation. Although highly unstable, this compound can be shown to inactivate the carboxylases from both Rhodospirillum rubrum and spinach rapidly and irreversibly. Inactivation follows pseudo first-order kinetics, shows rate saturation and is greatly reduced by saturating amounts of the competitive inhibitor, 2-carboxyribitol 1,5-bisphosphate. The incorporation of reagent, quantified by reducing the modified carboxylases with (/sup 3/H)NaBH/sub 4/, shows that inactivation results from the modification of approximately one residue per catalytic subunit of the Rhodospirillum rubrum enzyme and less than one residue per protomeric unit of the spinach enzyme.

  10. Regulation of bovine pyruvate carboxylase mRNA and promoter expression by thermal stress.

    PubMed

    White, H M; Koser, S L; Donkin, S S

    2012-09-01

    Pyruvate carboxylase (PC) catalyzes the rate-limiting step in gluconeogenesis from lactate and is a determinant of tricarboxylic acid cycle carbon flux. Bovine PC 5' untranslated region (UTR) mRNA variants are the products of a single PC gene containing 3 promoter regions (P3, P2, and P1, 5' to 3') that are responsive to physiological and nutritional stressors. The objective of this study was to determine the direct effects of thermal stress on PC mRNA and gene expression in bovine hepatocyte monolayer cultures, rat hepatoma (H4IIE) cells, and Madin-Darby bovine kidney epithelial (MDBK) cells. Hepatocytes were isolated from 3 Holstein bull calves and used to prepare monolayer cultures. Rat hepatoma cells and MDBK cells were obtained from American Type Culture Collection, Manassas, VA. Beginning 24 h after initial seeding, cells were subjected to either 37°C (control) or 42°C (thermal stress) for 24 h. Treatments were applied in triplicate in a minimum of 3 independent cell preparations. For bovine primary hepatocytes, endogenous expression of bovine PC mRNA increased (P < 0.1) with 24 h of thermal stress (1.31 vs. 2.79 ± 0.49, arbitrary units, control vs. thermal stress, respectively), but there was no change (P ≥ 0.1) in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) mRNA expression. Similarly, exposure of MDBK cells to thermal stress increased (P < 0.1) expression of bovine PC mRNA without altering (P ≥ 0.1) PEPCK-C mRNA expression. Conversely, there was no effect (P ≥ 0.1) of thermal stress on endogenous rat PC (0.47 vs. 0.30 ± 0.08, control vs. thermal stress) or PEPCK-C (1.61 vs. 1.20 ± 0.48, arbitrary units, control vs. thermal stress, respectively) mRNA expressions in H4IIE cells. To further investigate the regulation of PC, H4IIE cells were transiently transfected with bovine promoter-luciferase constructs containing either P1, P2, or P3, and exposed to thermal stress for 23 h. Activity of P1 was suppressed (P < 0.1) 5-fold, activity of P2

  11. Brønsted analysis reveals Lys218 as the carboxylase active site base that deprotonates vitamin K hydroquinone to initiate vitamin K-dependent protein carboxylation.

    PubMed

    Rishavy, Mark A; Hallgren, Kevin W; Yakubenko, Anna V; Shtofman, Rebecca L; Runge, Kurt W; Berkner, Kathleen L

    2006-11-07

    The vitamin K-dependent (VKD) carboxylase converts Glu's to carboxylated Glu's in VKD proteins to render them functional in a broad range of physiologies. The carboxylase uses vitamin K hydroquinone (KH(2)) epoxidation to drive Glu carboxylation, and one of its critical roles is to provide a catalytic base that deprotonates KH(2) to allow epoxidation. A long-standing model invoked Cys as the catalytic base but was ruled out by activity retention in a mutant where every Cys is substituted by Ala. Inhibitor analysis of the cysteine-less mutant suggested that the base is an activated amine [Rishavy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13732-13737], and in the present study, we used an evolutionary approach to identify candidate amines, which revealed His160, His287, His381, and Lys218. When mutational analysis was performed using an expression system lacking endogenous carboxylase, the His to Ala mutants all showed full epoxidase activity but K218A activity was not detectable. The addition of exogenous amines restored K218A activity while having little effect on wild type carboxylase, and pH studies indicated that rescue was dependent upon the basic form of the amine. Importantly, Brønsted analysis that measured the effect of amines with different pK(a) values showed that K218A activity rescue depended upon the basicity of the amine. The combined results provide strong evidence that Lys218 is the essential base that deprotonates KH(2) to initiate the reaction. The identification of this base is an important advance in defining the carboxylase active site and has implications regarding carboxylase membrane topology and the feedback mechanism by which the Glu substrate regulates KH(2) oxygenation.

  12. Kinetic Variance of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Isolated from Diverse Taxonomic Sources 1

    PubMed Central

    Kent, Samuel Sherrill; Tomany, Michael John

    1984-01-01

    Two dual label methods were used to investigate kinetic variability of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39). In addition to using [1-14C,5-3H]RuBP (method 1), we describe here the detailed assay with 14CO2 and [5-3H]RuBP (method 2), which generates [3H,14C]3-phosphoglyceric acid and unlabeled (noncontaminating) phosphoglycolate; the carboxylase/oxygenase activity ratio (vc/vo) is calculated from 3H/14C ratios of substrates and products. vc/vo was found to be a linear function of [CO2]/[O2], constant over a 4-minute assay interval, and invariant of the degree of enzyme activity. Accurately measurable vc/vo ratios range from approximately 0.3 to 6. The Km and Vmax of both enzymes may be determined as a composite constant, VcKo/VoKc. By method 2, the directly compared, relative values at 40 micromolar CO2 and 1240 micromolar O2 were: Spinacia oleracea (74), Chlorella pyrenoidosa (31), Plectonema boryanum (32), and Rhodospirillum rubrum (8). With method 1, the values for S. oleracea and R. rubrum were 75, and 9, respectively. Under tight experimental controls, the absolute value for S. oleracea was 69 ± 3. PMID:16663680

  13. Varying Photoperiod, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase and CO2 Uptake in Thalassiosira fluviatilis (Bacillariophyceae) 1

    PubMed Central

    Hobson, Louis A.; Morris, W. James.; Guest, Kathryn P.

    1985-01-01

    The purpose of this research was to test the hypothesis that acclimation of the unicellular marine alga, Thalassiosira fluviatilis Hustedt, to short photoperiods results in decreased cellular concentrations of ribulose 1,5-bisphosphate carboxylase/oxygenase and decreased rates of light-saturated CO2 uptake. Cells were acclimated to photoperiods of 6:18, 12:12, and 18:6 h:h light:dark, and concentrations of the large subunit of the enzyme and responses of CO2 uptake to varying irradiance were measured. Concentrations of the large subunit, which weighed approximately 50 kilodaltons, were conserved while rates of CO2 uptake under light saturation and limitation, and cellular contents of chlorophyll a increased as photoperiod decreased. Apparently, these cells acclimate to short photoperiods by increasing rates of CO2 uptake under saturating irradiances by increasing in vivo activation of ribulose 1,5-bisphosphate carboxylase/oxygenase. Also, chlorophyll-specific concentrations and specific activities of the enzyme appear to be lower and higher, respectively, in diatomaceous algae than in higher plants. Images Fig. 2 PMID:16664500

  14. The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development.

    PubMed

    Goedert, M; Jakes, R; Crowther, R A; Six, J; Lübke, U; Vandermeeren, M; Cras, P; Trojanowski, J Q; Lee, V M

    1993-06-01

    Tau is a neuronal phosphoprotein whose expression is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult brain, with the fetal isoform corresponding to the shortest of the adult isoforms. Phosphorylation of tau is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer disease, the six adult tau isoforms become abnormally phosphorylated and form the paired helical filament, the major fibrous component of the characteristic neurofibrillary lesions. We show here that Ser-202 (in the numbering of the longest human brain tau isoform) is a phosphorylation site that distinguishes fetal from adult tau and we identify it as one of the abnormal phosphorylation sites in Alzheimer disease. The abnormal phosphorylation of tau at Ser-202 in Alzheimer disease thus recapitulates normal phosphorylation during development.

  15. Effect of Selenium Deficiency on Phosphorylation of the AMPK Pathway in Rats.

    PubMed

    He, Shulan; Guo, Xiong; Tan, Wuhong; Su, Xiaohui; Li, Jiangping; Pan, Wang; Qiu, Hongyan

    2016-02-01

    Selenium is an important trace element for human health. Previous studies have raised concern that dietary selenium intake may change energy metabolism. AMP-activated protein kinase (AMPK) is a sensor of energy status that controls cellular energy homeostasis. We aimed to determine the effect of selenium on the phosphorylation of AMPK pathway between Se-deficient and normal Sprague-Dawley rats. Twenty-four weaning rats were fed either a Se-deficient diet (0.02 mg Se/kg) or a standard diet (0.18 mg Se/kg). After 109 days, total serum levels of non-esterified fatty acid and total amino acids were significantly higher and the serum insulin concentration was significantly lower in Se-deficient rats than in healthy controls. Selenium concentration and the activity of glutathione peroxidase (GPx) in myocardial tissue were significantly lower in Se-deficient rats. Importantly, mRNA levels of acetyl-CoA carboxylase beta (ACACB), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and protein levels of p-AMPKα were increased in the Se-deficient group compared to normal controls (p < 0.05). In conclusion, our results suggest that selenium deficiency induces changes in metabolic and molecular parameters involved in energy metabolism in the AMPK pathway.

  16. Enzymic and physicochemical characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from diploid and tetraploid cultivars of perennial ryegrass

    SciTech Connect

    Rejda, J.M.; Johal, S.; Chollet, R.

    1981-09-01

    Homogeneous preparations of ribulose 1,5-bisphosphate carboxylase/oxygenase were isolated from several diploid and tetraploid cultivars of perennial ryegrass by three different purification protocols. The apparent K/sub m/ values for substrate CO/sub 2/ were essentially identical for the fully CO/sub 2//Mg/sup 2 +/-activated diploid and tetraploid enzymes, as were the kinetics for deactivation and activation of the CO/sub 2//Mg/sup 2 +/-activated and -depleted carboxylases, respectively. Similarly, virtually indistinguishable electrophoretic properties were observed for both the native and dissociated diploid and tetraploid ryegrass proteins, including native and subunit molecular weights and the isoelectric points of the native proteins and the large and small subunit component polypeptides. The quantity of carboxylase protein or total soluble leaf protein did not differ significantly between the diploid and tetraploid cultivars. Contrary to a previous report, these results indicate that increased ploidy level has had essentially no effect on the quantity or enzymic and physicochemical properties of ribulosebisphosphate carboxylase/oxygenase in perennial ryegrass.

  17. Insulin stimulates the tyrosine phosphorylation of caveolin

    PubMed Central

    1995-01-01

    The specialized plasma membrane structures termed caveolae and the caveolar-coat protein caveolin are highly expressed in insulin- sensitive cells such as adipocytes and muscle. Stimulation of 3T3-L1 adipocytes with insulin significantly increased the tyrosine phosphorylation of caveolin and a 29-kD caveolin-associated protein in caveolin-enriched Triton-insoluble complexes. Maximal phosphorylation occurred within 5 min, and the levels of phosphorylation remained elevated for at least 30 min. The insulin-dose responses for the tyrosine phosphorylation of caveolin and the 29-kD caveolin-associated protein paralleled those for the phosphorylation of the insulin receptor. The stimulation of caveolin tyrosine phosphorylation was specific for insulin and was not observed with PDGF or EGF, although PDGF stimulated the tyrosine phosphorylation of the 29-kD caveolin- associated protein. Increased tyrosine phosphorylation of caveolin, its associated 29-kD protein, and a 60-kD protein was observed in an in vitro kinase assay after incubation of the caveolin-enriched Triton- insoluble complexes with Mg-ATP, suggesting the presence of an intrinsic tyrosine kinase in these complexes. These fractions contain only trace amounts of the activated insulin receptor. In addition, these complexes contain a 60-kD kinase detected in an in situ gel kinase assay and an approximately 60 kD protein that cross-reacts with an antibody against the Src-family kinase p59Fyn. Thus, the insulin- dependent tyrosine phosphorylation of caveolin represents a novel, insulin-specific signal transduction pathway that may involve activation of a tyrosine kinase downstream of the insulin receptor. PMID:7540611

  18. Control of light saturated photosynthesis: Concentration and activity of ribulose bisphosphate carboxylase. Final report, September 1, 1993--February 28, 1997

    SciTech Connect

    Geider, R.J. |

    1997-05-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is one of the most abundant enzymes on the planet and is responsible for catalysing the net fixation of CO{sub 2} into organic matter. It is central, therefore, to primary productivity in marine and terrestrial ecosystems. Rubisco is a large enzyme with low substrate affinity and low catalytic efficiency and is considered to limit the rate of light-saturated photosynthesis. This report summarizes research into the molecular basis of the regulation of phytoplankton photosynthesis. It describes experimental and theoretical studies of the role of Rubisco in regulating the photosynthetic rate of phytoplankton. It also describes the integration of a mechanistically based phytoplankton growth model into a description of primary productivity in the sea. This work was conducted as part of the Ocean Margins Program.

  19. Isolation of the catalytically competent small subunit of ribulose bisphosphate carboxylase/oxygenase from spinach under an extremely alkaline condition.

    PubMed

    Incharoensakdi, A; Takabe, T; Takabe, T; Akazawa, T

    1986-07-16

    A method for isolating the small subunit (B) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach leaf using an alkaline buffer (pH 11.2) in combination with sucrose gradient centrifugation is described. Although the yield of isolated subunit B (ca. 20%) was comparable to that previously described (ca. 25%) using the acid precipitation method [Andrews, T.J. and Lorimer, G.H. (1985) J. Biol. Chem. 260: 4632-4636], the isolated subunit B in this report suffered less denaturation (ca. 30%) as estimated from kinetic analysis of its reassembly with large subunit (A) derived from Aphanothece halophytica. Studies on the kinetic properties of the reassembled enzyme molecules suggested that spinach subunit B does not influence the affinity of the enzyme for substrate CO2. The catalytic core (A8) of spinach RuBisCO could not be isolated in the native form.

  20. The nature of L8 and L8S8 forms of ribulose bisphosphate carboxylase/oxygenase from Chromatium vinosum.

    PubMed

    Torres-Ruiz, J; McFadden, B A

    1987-04-01

    L8 and L8S8 forms of ribulose bisphosphate carboxylase/oxygenase (RubisCO) have been prepared from Chromatium vinosum by the extremely mild method of centrifugal fractionation. Only the L8S8 form is detectable in crude extracts of this organism. Both forms show immunological identify in double diffusion studies using antibody to L subunits of the L8S8 form. L subunits from both L8 and L8S8 enzymes are identical by the criteria of peptides observed after limited proteolysis and N-terminal sequence analysis. In addition, these subunits show regions of homology with L subunits from Rhodospirillum rubrum, Anacystis nidulans, and spinach. S subunits of the C. vinosum enzyme are completely homologous to those from A. nidulans and higher plants from the 18th through 25th residue, a stretch preceded in all cases by two basic amino acids.

  1. Activity ratios of ribulose-1,5-bisphosphate carboxylase accurately reflect carbamylation ratios. [Phaseolus vulgaris, Spinacla oleracea

    SciTech Connect

    Butz, N.D.; Sharkey, T.D. )

    1989-03-01

    Activity ratios and carbamylation ratios of ribulose-1,5-bisphosphate carboxylase (RuBPCase) were determined for leaves of Phaseolus vulgaris and Spinacia oleracea exposed to a variety of partial pressures of CO{sub 2} and O{sub 2} and photon flux densities (PFD). It was found that activity ratios accurately predicted carbamylation ratios except in extracts from leaves held in low PFD. In particular, it was confirmed that the loss of FuBPCase activity in low partial pressure of O{sub 2} and high PFD results from reduced carbamylation. Activity ratios of RuBPCase were lower than carbamylation ratios for Phaseolus leaves sampled in low PFD, presumably because of the presence of 2-carboxyarabinitol 1-phosphate. Spinacia leaves sampled in darkness also exhibited lower activity ratios than carbamylation ratios indicating that this species may also have an RuBPCase inhibitor even though carboxyarabinitol 1-phosphate has not been detected in this species in the past.

  2. In vitro reassembly of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase from fully denatured subunits.

    PubMed

    Yong, Zhen-Hua; Chen, Gen-Yun; Shi, Jiao-Nai; Xu, Da-Quan

    2006-10-01

    It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from fully denatured subunits in vitro in higher plant, because large subunit of fully denatured Rubisco is liable to precipitate when the denaturant is removed by common methods of direct dilution and one-step dialysis. In our experiment, the problem of precipitation was resolved by an improved gradual dialysis method, which gradually decreased the concentration of denaturant. However, fully denatured Rubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60 was added. The restored activity of reassembled Rubisco was approximately 8% of natural enzyme. The quantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassembly process. ATP and Mg2+ were unnecessary for in vitro reassembly of Rubisco, and Mg2+ inhibited the reassembly process. The reassembly was weakened when ATP, Mg2+ and K+ existed together in the reassembly process.

  3. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

    DOEpatents

    Houtz, R.L.

    1999-02-02

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS){sup {epsilon}}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 8 figs.

  4. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

    DOEpatents

    Houtz, R.L.

    1998-03-03

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) {epsilon}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 5 figs.

  5. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

    DOEpatents

    Houtz, Robert L.

    1999-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

  6. Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

    DOEpatents

    Houtz, Robert L.

    1998-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .epsilon.N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

  7. Synthesis of 7-oxo-dihydrospiro[indazole-5,4'-piperidine] acetyl-CoA carboxylase inhibitors.

    PubMed

    Bagley, Scott W; Southers, James A; Cabral, Shawn; Rose, Colin R; Bernhardson, David J; Edmonds, David J; Polivkova, Jana; Yang, Xiaojing; Kung, Daniel W; Griffith, David A; Bader, Scott J

    2012-02-03

    Synthesis of oxo-dihydrospiroindazole-based acetyl-CoA carboxylase (ACC) inhibitors is reported. The dihydrospiroindazoles were assembled in a regioselective manner in six steps from substituted hydrazines and protected 4-formylpiperidine. Enhanced regioselectivity in the condensation between a keto enamine and substituted hydrazines was observed when using toluene as the solvent, leading to selective formation of 1-substituted spiroindazoles. The 2-substituted spiroindazoles were formed selectively from alkyl hydrazones by ring closure with Vilsmeier reagent. The key step in the elaboration to the final products is the conversion of an intermediate olefin to the desired ketone through elimination of HBr from an O-methyl bromohydrin. This methodology enabled the synthesis of each desired regioisomer on 50-75 g scale with minimal purification. Acylation of the resultant spirocyclic amines provided potent ACC inhibitors.

  8. Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain.

    PubMed

    Weatherly, Stephanie C; Volrath, Sandra L; Elich, Tedd D

    2004-05-15

    Acetyl-CoA carboxylase (ACC) catalyses the first step in fatty-acid biosynthesis. Owing to its role in primary metabolism, ACC has been exploited as a commercial herbicide target and identified as a chemically validated fungicide target. In animals, ACC is also a key regulator of fat metabolism. This function has made ACC a prime target for the development of anti-obesity and anti-Type II diabetes therapeutics. Despite its economic importance, there is a lack of published information on recombinant expression of ACC. We report here the expression of enzymically active fungal (Ustilago maydis ) ACC in Escherichia coli. The recombinant enzyme exhibited Km values of 0.14+/-0.013 mM and 0.19+/-0.041 mM for acetyl-CoA and ATP respectively, which are comparable with those reported for the endogenous enzyme. The polyketide natural product soraphen is a potent inhibitor of the BC (biotin carboxylase) domain of endogenous fungal ACC. Similarly, recombinant ACC activity was inhibited by soraphen with a K(i) of 2.1+/-0.9 nM. A truncated BC domain that included amino acids 2-560 of the full-length protein was also expressed in E. coli. The isolated BC domain was expressed to higher levels, and was more stable than full-length ACC. Although incapable of enzymic turnover, the BC domain exhibited high-affinity soraphen binding (Kd 1.1+/-0.3 nM), demonstrating a native conformation. Additional BC domains from the phytopathogenic fungi Magnaporthe grisea and Phytophthora infestans were also cloned and expressed, and were shown to exhibit high-affinity soraphen binding. Together, these reagents will be useful for structural studies and assay development.

  9. Rubisco and PEP carboxylase responses to changing irradiance in a Brazilian Cerrado tree species, Qualea grandiflora Mart. (Vochysiaceae).

    PubMed

    Paulilo, M T; Besford, R T; Wilkins, D

    1994-02-01

    The activities of ribulose-1,5-bisphosphate carboxylase-oxygenase, Rubisco (E.C. 4.1.1.39) and phosphoenolpyruvate carboxylase, PEPc (E.C. 4.1.1.31), and concentrations of protein and chlorophyll were measured in extracts from cotyledons and first leaves of Qualea grandiflora Mart. (Vochysiaceae) seedlings after transfer from high-light (20 days at 320 micro mol m(-2) s(-1), PAR) to low-light (35 days at 120 micro mol m(-2) s(-1), PAR) conditions. When Tween 20 and glycerol were added to the extraction medium, Rubisco activities obtained for Qualea grandiflora were comparable to published values for several coniferous species and the broad-leaved species, Prunus avium L. Stella, grown in a similar light environment. Rubisco activity in cotyledons of Q. grandiflora grown in high light for 20 days and then transferred to low light for a further 35 days was similar to the activity in cotyledons of plants grown continuously in high light. However, the first leaf above the cotyledons showed a greater response to the change in irradiance; in high light, Rubisco activity of the first leaf was 1.8 times higher on a fresh weight basis and 2.7 times higher on an area basis than that of leaves transferred from high to low light. Fresh weight and chlorophyll concentration expressed on a unit leaf area basis were also higher in the high-light treatment. These responses to irradiance are indicative of a species adapted to growth in an unshaded habitat. The PEPc activity in leaves was 15% of Rubisco activity, which is typical of species with a C(3) photosynthetic pathway. The relatively slow growth rate of Q. grandiflora observed in these experiments could not be attributed to a low carboxylation capacity per unit leaf area.

  10. Cloning and characterization of pyruvate carboxylase gene responsible for calcium malate overproduction in Penicillium viticola 152 and its expression analysis.

    PubMed

    Khan, Ibrar; Qayyum, Sadia; Ahmed, Shehzad; Maqbool, Farhana; Tauseef, Isfahan; Haleem, Kashif Syed; Chi, Zhen-Ming

    2017-03-20

    In this study, a pyruvate carboxylase gene (PYC) from a marine fungus Penicillium viticola 152 isolated from marine algae was cloned and characterized by using Genome Walking method. An open reading frame (ORF) of The PYC gene (accession number: KM593097) had 3582bp encoding 1193 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 131.2757kDa. A putative promoter (intronless) of the gene was located at -666bp and contained a TATA box, several CAAT boxes, the 5'-SYGGRG-3' and a 5'-HGATAR-3' sequences. A consensus polyadenylation site (AATAAA) was also observed at +10bp downstream of the ORF. The protein deduced from the PYC gene had no signal peptide, was a homotetramer (4), and had the four functional domains. Furthermore, PYC protein also had three potential N-linked glycosylation sites, among them, -N-S-T-I- at 36 amino acid, -N-G-T-V- at 237 amino acid, and -N-G-S-S- at 517 amino acid were the most possible N-glycosylation sites. After expression of the PYC gene of P. viticola 152 in medium supplemented with CSL and biotin, it was found that the specific pyruvate carboxylase activity in MA production medium supplemented with CSL was much higher (0.5U/mg) than in MA medium supplemented with biotin (0.3U/mg), suggesting that optimal concentration of CSL is required for increased expression of the PYC gene, which is responsible for high level production of malic acid in P. viticola 152 strain.

  11. Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue

    SciTech Connect

    Smith, H.B.; Hartman, F.C. )

    1991-05-28

    Ribulosebisphosphate carboxylase/oxygenase is reversibly activated by the reaction of Co{sub 2} with a specific lysyl residue to form a carbamate that coordinates an essential Mg{sup 2+} cation. Surprisingly, the Lys191{yields}Cys mutant protein, in the presence of Co{sub 2} and Mg{sup 2+} exhibits tight binding of the reaction intermediate analogue 2-carboxyarabinitol bisphosphate a property normally equated with effective coordination of the Mg{sup 2+} by the carbamate. Catalytic ineptness of the Cys191 mutant protein, despite its ability to coordinate Mg{sup 2+} properly, might be due to the absence of the carbamate nitrogen. To investigate this possibility, the authors have evaluated the ability of exogenous amines to restore catalytic activity to the mutant protein. Significantly, the Cys191 protein manifests ribulose bisphosphate dependent fixation of {sup 14}CO{sub 2} when incubated with aminomethanewsulfonate but not ethanesulfonate. This novel activity reflects a K{sub m} value for ribulose bisphosphate which is not markedly perturbed relative to wild-type enzyme, a K{sub m} for Mg{sup 2+} which is in fact decreased 10-fold, and rate saturation with respect to aminomethanesulfonate. Chromatographic and spectrophotometric analyses reveal the product of CO{sub 2} fixation to be D-3-phosphoglycerate while turnover of (1-{sup 3}H)ribulose bisphosphate into ({sup 3}H)phosphoglycolate confirms oxygenase activity. The authors conclude that aminomethanesulfonate restored ribulosebisphosphate carboxylase/oxygenase activities to the Cys191 mutant protein by providing a nitrogenous function which satisfies a catalytic demand normally met by the carbamate nitrogen of Lys191.

  12. Examining site-specific GPCR phosphorylation.

    PubMed

    Butcher, Adrian J; Tobin, Andrew B; Kong, Kok Choi

    2011-01-01

    Phosphorylation of G protein-coupled receptors (GPCRs) is one of the most prominent post-translation modifications mediated by agonist stimulation. This process has been shown to result not only in receptor desensitisation but also, via the recruitment of arrestin adaptor proteins, to promote receptor coupling to numerous signalling pathways. Furthermore, there is now a growing body of evidence suggesting that GPCRs may employ phosphorylation as a mechanism to regulate their cell-type-specific signalling, hence generating tissue-specific functions. These advances have resulted partly from improved methods used in the determination of phospho-acceptor sites on GPCRs and improved analysis of the consequences of phosphorylation. This chapter aims to describe the methods used in our laboratory for the investigation of site-specific phosphorylation of the M₃-muscarinic receptor. These methods could easily be applied in the study of other receptors.

  13. Protein phosphorylation in neurodegeneration: friend or foe?

    PubMed Central

    Tenreiro, Sandra; Eckermann, Katrin; Outeiro, Tiago F.

    2014-01-01

    Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and fronto-temporal dementia (FTD). In these disorders, the misfolding and aggregation of specific proteins occurs alongside neuronal degeneration in somewhat specific brain areas, depending on the disorder and the stage of the disease. However, we still do not fully understand the mechanisms governing protein aggregation, and whether this constitutes a protective or detrimental process. In PD, alpha-synuclein (aSyn) forms protein aggregates, known as Lewy bodies, and is phosphorylated at serine 129. Other residues have also been shown to be phosphorylated, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. In AD and in FTD, hyperphosphorylation of tau protein causes its misfolding and aggregation. Again, our understanding of the precise consequences of tau phosphorylation in the biology and pathophysiology of the protein is still limited. Through the use of a variety of model organisms and technical approaches, we are now gaining stronger insight into the effects of phosphorylation in the behavior of these proteins. In this review, we cover recent findings in the field and discuss how targeting phosphorylation events might be used for therapeutic intervention in these devastating diseases of the nervous system. PMID:24860424

  14. Long-term dynamics of multisite phosphorylation

    PubMed Central

    Rubinstein, Boris Y.; Mattingly, Henry H.; Berezhkovskii, Alexander M.; Shvartsman, Stanislav Y.

    2016-01-01

    Multisite phosphorylation cycles are ubiquitous in cell regulation systems and are studied at multiple levels of complexity, from molecules to organisms, with the ultimate goal of establishing predictive understanding of the effects of genetic and pharmacological perturbations of protein phosphorylation in vivo. Achieving this goal is essentially impossible without mathematical models, which provide a systematic framework for exploring dynamic interactions of multiple network components. Most of the models studied to date do not discriminate between the distinct partially phosphorylated forms and focus on two limiting reaction regimes, distributive and processive, which differ in the number of enzyme–substrate binding events needed for complete phosphorylation or dephosphorylation. Here we use a minimal model of extracellular signal-related kinase regulation to explore the dynamics of a reaction network that includes all essential phosphorylation forms and arbitrary levels of reaction processivity. In addition to bistability, which has been studied extensively in distributive mechanisms, this network can generate periodic oscillations. Both bistability and oscillations can be realized at high levels of reaction processivity. Our work provides a general framework for systematic analysis of dynamics in multisite phosphorylation systems. PMID:27226482

  15. Compartment-Specific Phosphorylation of Squid Neurofilaments.

    PubMed

    Grant, Philip; Pant, Harish C

    2016-01-01

    Studies of the giant axon and synapse of third-order neurons in the squid stellate ganglion have provided a vast literature on neuronal physiology and axon transport. Large neuronal size also lends itself to comparative biochemical studies of cell body versus axon. These have focused on the regulation of synthesis, assembly, posttranslational modification and function of neuronal cytoskeletal proteins (microtubules (MTs) and neurofilaments (NFs)), the predominant proteins in axoplasm. These contribute to axonal organization, stability, transport, and impulse transmission responsible for rapid contractions of mantle muscles underlying jet propulsion. Studies of vertebrate NFs have established an extensive literature on NF structure, organization, and function; studies of squid NFs, however, have made it possible to compare compartment-specific regulation of NF synthesis, assembly, and function in soma versus axoplasm. Since NFs contain over 100 eligible sites for phosphorylation by protein kinases, the compartment-specific patterns of phosphorylation have been a primary focus of biochemical studies. We have learned that NF phosphorylation is tightly compartmentalized; extensive phosphorylation occurs only in the axonal compartment in squid and in vertebrate neurons. This extensive phosphorylation plays a key role in organizing NFs, in association with microtubules (MTs), into a stable, dynamic functional lattice that supports axon growth, diameter, impulse transmission, and synaptic activity. To understand how cytoskeletal phosphorylation is topographically regulated, the kinases and phosphatases, bound to NFs isolated from cell bodies and axoplasm, have also been studied.

  16. Phosphorylation of human skeletal muscle myosin

    SciTech Connect

    Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

    1986-03-01

    Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30/sup 0/C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with (/sup 30/P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ.

  17. Protein phosphorylation: Localization in regenerating optic axons

    SciTech Connect

    Larrivee, D. )

    1990-09-01

    A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

  18. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    SciTech Connect

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.

  19. Phosphorylation of Recombinant Tristetraprolin in Vitro

    PubMed Central

    Cao, Heping; Lin, Rui

    2009-01-01

    Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some proinflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of proinflammatory cytokines. TTP gene expression is induced by various factors including insulin, cinnamon, and green tea extracts. Previous studies have shown that TTP is highly phosphorylated in vivo and multiple phosphorylation sites are identified in human TTP. This study evaluated the potential protein kinases that could phosphorylate recombinant TTP in vitro. Motif scanning suggested that TTP was a potential substrate for various kinases. SDS-PAGE showed that in vitro phosphorylation of TTP with p42 and p38 MAP kinases resulted in visible electrophoretic mobility shift of TTP to higher molecular masses. Autoradiography showed that TTP was phosphorylated in vitro by GSK3b, PKA, PKB, PKC, but not Cdc2, in addition to p42, p38, and JNK. These results demonstrate that TTP is a substrate for a number of protein kinases in vitro. PMID:18071886

  20. Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation

    PubMed Central

    Rigatti, Marc; Le, Andrew V.; Gerber, Claire; Moraru, Ion I.; Dodge-Kafka, Kimberly L.

    2016-01-01

    Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca2+ cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca2+ re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and Δ14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7δ/γ and display reduced phosphorylation in vitro. This finding implicates the AKAP7δ/γ-PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7δ/γ-PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100–200nM) to regulate the phosphorylation of large quantities of PLB (50µM). Our results confirm that AKAP7γ/δ binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100–200 fold lower concentrations. PMID:26027516

  1. Phosphorylation of RACK1 in plants

    SciTech Connect

    Chen, Jay -Gui

    2015-08-31

    Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades. RACK1 has been shown to regulate hormonal signaling, stress responses and multiple processes of growth and development in plants. However, little is known about the molecular mechanism underlying these regulations. Recently, it has been demonstrated that Arabidopsis RACK1 is phosphorylated by an atypical serine/threonine protein kinase, WITH NO LYSINE 8 (WNK8). Furthermore, RACK1 phosphorylation by WNK8 negatively regulates RACK1 function by influencing its protein stability. In conclusion, these findings promote a new regulatory system in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation.

  2. Phosphorylation mechanisms in dopamine transporter regulation.

    PubMed

    Foster, James D; Vaughan, Roxanne A

    2016-11-09

    The dopamine transporter (DAT) is a plasma membrane phosphoprotein that actively translocates extracellular dopamine (DA) into presynaptic neurons. The transporter is the primary mechanism for control of DA levels and subsequent neurotransmission, and is the target for abused and therapeutic drugs that exert their effects by suppressing reuptake. The transport capacity of DAT is acutely regulated by signaling systems and drug exposure, providing neurons the ability to fine-tune DA clearance in response to specific conditions. Kinase pathways play major roles in these mechanisms, and this review summarizes the current status of DAT phosphorylation characteristics and the evidence linking transporter phosphorylation to control of reuptake and other functions. Greater understanding of these processes may aid in elucidation of their possible contributions to DA disease states and suggest specific phosphorylation sites as targets for therapeutic manipulation of reuptake.

  3. Phosphorylation of RACK1 in plants

    DOE PAGES

    Chen, Jay -Gui

    2015-08-31

    Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades. RACK1 has been shown to regulate hormonal signaling, stress responses and multiple processes of growth and development in plants. However, little is known about the molecular mechanism underlying these regulations. Recently, it has been demonstrated that Arabidopsis RACK1 is phosphorylated by an atypical serine/threonine protein kinase, WITH NO LYSINE 8 (WNK8). Furthermore, RACK1 phosphorylation by WNK8 negatively regulates RACK1 function by influencing its protein stability. In conclusion, these findings promote a new regulatory systemmore » in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation.« less

  4. High substrate specificity factor ribulose bisphosphate carboxylase/oxygenase from eukaryotic marine algae and properties of recombinant cyanobacterial RubiSCO containing "algal" residue modifications.

    PubMed

    Read, B A; Tabita, F R

    1994-07-01

    Marine algae play an important role in removing carbon dioxide from the atmosphere. In this investigation, we have determined the substrate specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase from several marine chromophytic and rhodophytic algae. The enzymes were purified to homogeneity and all possessed significantly higher substrate specificity factors than the enzymes from terrestrial plants, green algae, or bacteria. There are substantial differences in the sequence in a helix 6 of the large subunit of these enzymes, which is intriguing since residues of this region had been previously shown to influence the ability of ribulose bisphosphate carboxylase to discriminate between CO2 and O2, presumably by influencing the adjacent flexible loop 6 region. Sequence divergence at this and other key regions might contribute to the substantial differences in the substrate specificity factor of the chromophyte/rhodophyte enzyme. Initial studies on probing the basis for the high substrate specificity factor employed single amino acid substitutions in the recombinant cyanobacterial ribulose bisphosphate carboxylase. Residues in the vicinity of loop 6 were changed to reflect the corresponding residues in the chromophyte/rhodophyte large subunit. Some changes in the substrate specificity factor were noted, as were alterations in other important kinetic parameters. Since marine algae show little evidence of photorespiratory metabolism, the high substrate specificity of ribulose bisphosphate carboxylase is consistent with the physiology of these organisms. The results of this study provide further evidence that the properties of this enzyme may evolve or change according to the environment in which the host organism is found.

  5. AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

    SciTech Connect

    Adam, Tasneem; Opie, Lionel H.; Essop, M. Faadiel

    2010-07-30

    Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional level.

  6. Biochemical characterization of a Rhizobium etli monovalent cation-stimulated acyl-coenzyme A carboxylase with a high substrate specificity constant for propionyl-coenzyme A.

    PubMed

    Dunn, Michael F; Araíza, Gisela; Mora, Jaime

    2004-02-01

    Biotin has a profound effect on the metabolism of rhizobia. It is reported here that the activities of the biotin-dependent enzymes acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) and propionyl-coenzyme A carboxylase (PCC; EC 6.4.1.3) are present in all species of the five genera comprising the Rhizobiaceae which were examined. Evidence is presented that the ACC and PCC activities detectable in Rhizobium etli extracts are catalysed by a single acyl-coenzyme A carboxylase. The enzyme from R. etli strain 12-53 was purified 478-fold and displayed its highest activity with propionyl-CoA as substrate, with apparent K(m) and V(max) values of 0.064 mM and 2885 nmol min(-1) (mg protein)(-1), respectively. The enzyme carboxylated acetyl-CoA and butyryl-CoA with apparent K(m) values of 0.392 and 0.144 mM, respectively, and V(max) values of 423 and 268 nmol min(-1) (mg protein)(-1), respectively. K(+), or Cs(+) markedly activated the enzyme, which was essentially inactive in their absence. Electrophoretic analysis indicated that the acyl-CoA carboxylase was composed of a 74 kDa biotin-containing alpha subunit and a 45 kDa biotin-free beta subunit, and gel chromatography indicated a total molecular mass of 620 000 Da. The strong kinetic preference of the enzyme for propionyl-CoA is consistent with its participation in an anaplerotic pathway utilizing this substrate.

  7. Identification of dual Acetyl-CoA carboxylases 1 and 2 inhibitors by pharmacophore based virtual screening and molecular docking approach.

    PubMed

    Bhadauriya, Anuseema; Dhoke, Gaurao V; Gangwal, Rahul P; Damre, Mangesh V; Sangamwar, Abhay T

    2013-02-01

    Acetyl-CoA carboxylase (ACC) is a crucial metabolic enzyme that plays a vital role in obesity-induced type 2 diabetes and fatty acid metabolism. To identify dual inhibitors of Acetyl-CoA carboxylase1 and Acetyl-CoA carboxylase2, a pharmacophore modelling approach has been employed. The best HypoGen pharmacophore model for ACC2 inhibitors (Hypo1_ACC2) consists of one hydrogen bond acceptor, one hydrophobic aliphatic and one hydrophobic aromatic feature, whereas the best pharmacophore (Hypo1_ACC1) for ACC1 consists of one additional hydrogen-bond donor (HBD) features. The best pharmacophore hypotheses were validated by various methods such as test set, decoy set and Cat-Scramble methodology. The validated pharmacophore models were used to screen several small-molecule databases, including Specs, NCI, ChemDiv and Natural product databases to identify the potential dual ACC inhibitors. The virtual hits were then subjected to several filters such as estimated [Formula: see text] value, quantitative estimation of drug-likeness and molecular docking analysis. Finally, three novel compounds with diverse scaffolds were selected as potential starting points for the design of novel dual ACC inhibitors.

  8. Site-specific mutations in a loop region of the C-terminal domain of the large subunit of ribulose bisphosphate carboxylase/oxygenase that influence substrate partitioning.

    PubMed

    Gutteridge, S; Rhoades, D F; Herrmann, C

    1993-04-15

    Amino acids composing a flexible loop (loop 6) of the eight-stranded barrel domain of the L-subunit of Synechococcus ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) involved in reaction intermediate stabilization have been modified by site-specific mutagenesis. Changes at positions both distant and within the active site affect overall catalysis and substrate partitioning. Most significantly, replacement of the active site Lys (Lys-334) with Arg at the apex of the loop almost completely suppressed the carboxylase activity of the enzyme relative to oxygenation, with only a modest reduction in overall catalysis. Val-331 and Thr-342, more distant from the active site but with interacting side chains, were changed to larger and smaller residues with differential effects on both turnover and substrate partitioning. Substitution of the loop with the sequence found in more efficient carboxylases only increased partitioning marginally when accompanied by alterations in the C-terminal tail of the L-subunit that interacts with the loop. Generally, modifications to the loop composition also affected enediol formation, the first step of catalysis, suggesting that the geometry and hence flexibility of this segment affect more than just stabilization of the intermediates immediately following reaction with CO2 or O2.

  9. Resistance to herbicides caused by single amino acid mutations in acetyl-CoA carboxylase in resistant populations of grassy weeds.

    PubMed

    Jang, SoRi; Marjanovic, Jasmina; Gornicki, Piotr

    2013-03-01

    Eleven spontaneous mutations of acetyl-CoA carboxylase have been identified in many herbicide-resistant populations of 42 species of grassy weeds, hampering application of aryloxyphenoxypropionate, cyclohexadione and phenylpyrazoline herbicides in agriculture. IC(50) shifts (resistance indices) caused by herbicide-resistant mutations were determined using a recombinant yeast system that allows comparison of the effects of single amino acid mutations in the same biochemical background, avoiding the complexity inherent in the in planta experiments. The effect of six mutations on the sensitivity of acetyl-CoA carboxylase to nine herbicides representing the three chemical classes was studied. A combination of partially overlapping binding sites of the three classes of herbicides and the structure of their variable parts explains cross-resistance among and between the three classes of inhibitors, as well as differences in their specificity. Some degree of resistance was detected for 51 of 54 herbicide/mutation combinations. Introduction of new herbicides targeting acetyl-CoA carboxylase will depend on their ability to overcome the high degree of cross-resistance already existing in weed populations.

  10. Susceptibilities of Different Test Systems from Maize (Zea mays), Poa annua, and Festuca rubra to Herbicides That Inhibit the Enzyme Acetyl-Coenzyme A Carboxylase

    PubMed

    Herbert; Cole; Pallett; Harwood

    1996-06-01

    The susceptibilities of maize (Zea mays cv. Champ) and two graminicide-resistant grass species, Poa annua (annual meadow grass) and Festuca rubra (red fescue), to two aryloxyphenoxypropionates (quizalofop and fluazifop) and a cyclohexanedione (sethoxydim) graminicide were evaluated in leaf blades and isolated chloroplasts, and by assaying acetyl-coenzyme A carboxylase (ACCase) in desalted leaf homogenates. The graminicide resistance of P. annua and F. rubra appeared to be at the level of ACCase. Festuca rubra ACCase was highly insensitive and P. annua ACCase was partially insensitive to the graminicides that were tested. Fatty acid synthesis in isolated maize chloroplasts was more susceptible to inhibition than was ACCase activity from whole leaves. There was a smaller difference in graminicide sensitivity between these two test systems in P. annua. The developmental pattern of ACCase specific activity and its inhibition by quizalofop was measured in maize and P. annua leaf blades. There was an age-dependent increase in the sensitivity of maize leaf ACCase activity to inhibition by quizalofop. Together with the greater susceptibility of chloroplasts compared with leaf homogenates this could imply that a graminicide-insensitive (extrachloroplastic) ACCase isoform is less highly expressed in older leaves. Poa annua ACCase did not significantly alter in sensitivity as leaves aged, consistent with the smaller difference in the level of inhibition between chloroplasts and leaf homogenates in this species. A small pyruvate carboxylase activity was detected in maize leaves after 9 days. By 38 days, when leaves were senescing, pyruvate carboxylase activity predominated over ACCase.

  11. Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation

    PubMed Central

    Wang, Yifan; Zhang, Yanchong; Hu, Wen; Xie, Shutao; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2015-01-01

    Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3β (GSK-3β) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied. PMID:26511732

  12. Dominant mutations causing alterations in acetyl-coenzyme A carboxylase confer tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides in maize.

    PubMed Central

    Parker, W B; Marshall, L C; Burton, J D; Somers, D A; Wyse, D L; Gronwald, J W; Gengenbach, B G

    1990-01-01

    A partially dominant mutation exhibiting increased tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides was isolated by exposing susceptible maize (Zea mays) tissue cultures to increasingly inhibitory concentrations of sethoxydim (a cyclohexanedione). The selected tissue culture (S2) was greater than 40-fold more tolerant to sethoxydim and 20-fold more tolerant to haloxyfop (an aryloxyphenoxypropionate) than the nonselected wild-type tissue culture. Regenerated S2 plants were heterozygous for the mutant allele and exhibited a high-level, but not complete, tolerance to both herbicides. Homozygous mutant families derived by self-pollinating the regenerated S2 plants exhibited no injury after treatment with 0.8 kg of sethoxydim per ha, which was greater than 16-fold the rate lethal to wild-type plants. Acetyl-coenzyme A carboxylase (ACCase; EC 6.4.1.2) is the target enzyme of cyclohexanedione and aryloxyphenoxypropionate herbicides. ACCase activities of the nonselected wild-type and homozygous mutant seedlings were similar in the absence of herbicide. ACCase activity from homozygous tolerant plants required greater than 100-fold more sethoxydim and 16-fold more haloxyfop for 50% inhibition than ACCase from wild-type plants. These results indicate that tolerance to sethoxydim and haloxyfop is controlled by a partially dominant nuclear mutation encoding a herbicide-insensitive alteration in maize ACCase. Images PMID:1976254

  13. Multiple isoforms of phosphoenolpyruvate carboxylase in the Orchidaceae (subtribe Oncidiinae): implications for the evolution of crassulacean acid metabolism.

    PubMed

    Silvera, Katia; Winter, Klaus; Rodriguez, B Leticia; Albion, Rebecca L; Cushman, John C

    2014-07-01

    Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and subsequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of transcriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and photosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independent origins.

  14. Temperature Responses of C4 Photosynthesis: Biochemical Analysis of Rubisco, Phosphoenolpyruvate Carboxylase, and Carbonic Anhydrase in Setaria viridis1[OPEN

    PubMed Central

    Boyd, Ryan A.; Gandin, Anthony; Cousins, Asaph B.

    2015-01-01

    The photosynthetic assimilation of CO2 in C4 plants is potentially limited by the enzymatic rates of Rubisco, phosphoenolpyruvate carboxylase (PEPc), and carbonic anhydrase (CA). Therefore, the activity and kinetic properties of these enzymes are needed to accurately parameterize C4 biochemical models of leaf CO2 exchange in response to changes in CO2 availability and temperature. There are currently no published temperature responses of both Rubisco carboxylation and oxygenation kinetics from a C4 plant, nor are there known measurements of the temperature dependency of the PEPc Michaelis-Menten constant for its substrate HCO3−, and there is little information on the temperature response of plant CA activity. Here, we used membrane inlet mass spectrometry to measure the temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc carboxylation kinetics, and the activity and first-order rate constant for the CA hydration reaction from 10°C to 40°C using crude leaf extracts from the C4 plant Setaria viridis. The temperature dependencies of Rubisco, PEPc, and CA kinetic parameters are provided. These findings describe a new method for the investigation of PEPc kinetics, suggest an HCO3− limitation imposed by CA, and show similarities between the Rubisco temperature responses of previously measured C3 species and the C4 plant S. viridis. PMID:26373659

  15. Reversible inactivation and characterization of purified inactivated form I ribulose 1,5-bisphosphate carboxylase/oxygenase of Rhodobacter sphaeroides.

    PubMed

    Wang, X; Tabita, F R

    1992-06-01

    Form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Rhodobacter sphaeroides is inactivated upon the addition of organic acids to photolithoautotrophically grown cultures. Activity recovers after the dissipation of the organic acid from the culture. The inactivation process depends on both the concentration of the organic compound and the nitrogen status of the cells. The inactivated RubisCO has been purified and was shown to exhibit mobility on both nondenaturing and sodium dodecyl sulfate gels different from that of the active enzyme prepared from cells not treated with organic acids. However, the Michaelis constants for ribulose 1,5-bisphosphate and CO2 or O2 were not dramatically altered. Purified inactivated RubisCO could be activated in vitro by increasing the temperature or the levels of Mg(II), and this activation was accompanied by changes in the electrophoretic mobility of the protein. When foreign bacterial RubisCO genes were expressed in an R. sphaeroides host strain lacking the ability to synthesize endogenous RubisCO, only slight inactivation of RubisCO activity was attained.

  16. Closely related form I ribulose bisphosphate carboxylase/oxygenase molecules that possess different CO2/O2 substrate specificities.

    PubMed

    Horken, K M; Tabita, F R

    1999-01-15

    The deduced primary sequence (cbbL and cbbS) of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (rubisco) from Bradyrhizobium japonicum places this enzyme within the Type IC subgroup of red-like rubisco enzymes. In addition, B. japonicum appears to organize most of the structural genes of the Calvin-Benson-Bassham (CBB) pathway in at least one major operon. Functional expression and characterization of the B. japonicum and Xanthobacter flavus enzymes from this group revealed that these molecules exhibit diverse kinetic properties despite their relatively high degree of sequence relatedness. Of prime importance was the fact that these closely related enzymes exhibited CO2 and O2 substrate specificities that varied from relatively low values [tau = (VcKo)/(VoKc) = 45] to values that approximated those obtained for higher plants (tau = 75). These results, combined with the metabolic and genetic versatility of the organisms from which these enzymes were derived, suggest a potential rich resource for future biological selection and structure-function studies aimed at elucidating structural features that govern key enzymological properties of rubisco.

  17. Effect of mutation of lysine-128 of the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans.

    PubMed

    Bainbridge, G; Anralojc, P J; Madgwick, P J; Pitts, J E; Parry, M A

    1998-12-01

    The contribution of lysine-128 within the active site of Anacystis nidulans d-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was investigated by the characterization of mutants in which lysine-128 was replaced with arginine, glycine, glutamine, histidine or aspartic acid. Mutated genes encoding the Rubisco large subunit were expressed in Escherichia coli and the resultant polypeptides assembled into active complexes. All of the mutant enzymes had a lower affinity for ribulose 1,5-bisphosphate (RuBP) and lower rates of carboxylation. Substitution of lysine-128 with glutamine, histidine or aspartic acid decreased the specificity factor and led to the production of an additional monophosphate reaction product. We show that this product results from the loss of the phosphate from C-1 of RuBP, most probably by beta-elimination from the 2,3-enediolate derivative of RuBP. The results confirm that lysine-128 is important in determining the position of the essential epsilon-amino group of lysine-334 within the active site and in loop dynamics. This further demonstrates that residues remote from the active site can be manipulated to modify catalytic function.

  18. Constitutive and dark-induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase enhances stomatal opening and photosynthetic performance of Arabidopsis thaliana.

    PubMed

    Kebeish, Rashad; Niessen, Markus; Oksaksin, Mehtap; Blume, Christian; Peterhaensel, Christoph

    2012-02-01

    The effect of constitutive and dark-induced expression of Solanum tuberosum phosphoenolpyruvate carboxylase (PEPC) on the opening state of stomata and photosynthetic performance in Arabidopsis thaliana plants was studied. Transcript accumulation analyses of the A. thaliana dark-induced (Din10 and Din6) and the Pisum sativum asparagine synthetase 2 promoters (Asn2) in transiently transformed tobacco leaves showed that Din10 promoter induced more DsRed accumulation in the dark compared to the other din genes. Overexpression of PEPC under the control of the constitutive enhanced CaMV 35S (p35SS) and dark-induced Din10 promoter in stably transformed A. thaliana plants increased the number of opened stomata in dark adapted leaves. Gas exchange measurements using A. thaliana plants transgenic for p35SS-PEPC and Din10-PEPC revealed a marked increase in stomatal conductance, transpiration, and dark respiration rates measured in the dark compared to wild-type plants. Moreover, measurement of CO(2) assimilation rates at different external CO(2) concentrations (C(a) ) and different light intensities shows an increase in the CO(2) assimilation rates in transgenic Arabidopsis lines compared to wild-type plants. This is considered as first step towards transferring the aspects of Crassulacean acid metabolism-like photosynthetic mechanism into C3 plants.

  19. Diversity of the ribulose bisphosphate carboxylase/oxygenase form I gene (rbcL) in natural phytoplankton communities.

    PubMed Central

    Pichard, S L; Campbell, L; Paul, J H

    1997-01-01

    The phytoplankton of the world's oceans play an integral part in global carbon cycling and food webs by conversion of carbon dioxide into organic carbon. They accomplish this task through the action of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Here we have investigated the phylogenetic diversity in the form I rbcL locus in natural phytoplankton communities of the open ocean and representative clones of marine autotrophic picoplankton by mRNA or DNA amplification and sequencing of a 480 to 483 bp internal fragment of this gene. Five gene sequences were recovered from nucleic acids of natural phytoplankton communities of the Gulf of Mexico. The rbcL genes of two Prochlorococcus isolates and one Synechococcus strain (WH8007) were also sequenced. Sequences were aligned with the database of rbcL genes and subjected to both neighbor-joining and parsimony analyses. The five sequences from the natural phytoplankton community spanned nearly the entire diversity of characterized form I rbcL genes, with some sequences closely related to isolates such as Synechococcus and Prochlorococcus (forms IA and I) and prymnesiophyte algae (form ID), while other sequences were deeply rooted. Unexpectedly, the deep euphotic zone contained an organism that possesses a transcriptionally active rbcL gene closely related to that of a recently characterized manganese-oxidizing bacterium, suggesting that such chemoautotrophs may contribute to the diversity of carbon-fixing organisms in the marine euphotic zone. PMID:9293012

  20. Molecular basis for the inhibition of the carboxyltransferase domain of acetyl-coenzyme-A carboxylase by haloxyfop and diclofop

    PubMed Central

    Zhang, Hailong; Tweel, Benjamin; Tong, Liang

    2004-01-01

    Acetyl-CoA carboxylases (ACCs) are crucial for the metabolism of fatty acids, making these enzymes important targets for the development of therapeutics against obesity, diabetes, and other diseases. The carboxyltransferase (CT) domain of ACC is the site of action of commercial herbicides, such as haloxyfop, diclofop, and sethoxydim. We have determined the crystal structures at up to 2.5-Å resolution of the CT domain of yeast ACC in complex with the herbicide haloxyfop or diclofop. The inhibitors are bound in the active site, at the interface of the dimer of the CT domain. Unexpectedly, inhibitor binding requires large conformational changes for several residues in this interface, which create a highly conserved hydrophobic pocket that extends deeply into the core of the dimer. Two residues that affect herbicide sensitivity are located in this binding site, and mutation of these residues disrupts the structure of the domain. Other residues in the binding site are strictly conserved among the CT domains. PMID:15079078

  1. Acetyl-CoA carboxylase inhibition by ND-630 reduces hepatic steatosis, improves insulin sensitivity, and modulates dyslipidemia in rats

    PubMed Central

    Harriman, Geraldine; Greenwood, Jeremy; Bhat, Sathesh; Huang, Xinyi; Wang, Ruiying; Paul, Debamita; Tong, Liang; Saha, Asish K.; Westlin, William F.; Kapeller, Rosana; Harwood, H. James

    2016-01-01

    Simultaneous inhibition of the acetyl-CoA carboxylase (ACC) isozymes ACC1 and ACC2 results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation and may favorably affect the morbidity and mortality associated with obesity, diabetes, and fatty liver disease. Using structure-based drug design, we have identified a series of potent allosteric protein–protein interaction inhibitors, exemplified by ND-630, that interact within the ACC phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit the enzymatic activity of both ACC isozymes, reduce fatty acid synthesis and stimulate fatty acid oxidation in cultured cells and in animals, and exhibit favorable drug-like properties. When administered chronically to rats with diet-induced obesity, ND-630 reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia. When administered chronically to Zucker diabetic fatty rats, ND-630 reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c (0.9% reduction). Together, these data suggest that ACC inhibition by representatives of this series may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes mellitus, and fatty liver disease. PMID:26976583

  2. Photosynthetic and Other Phosphoenolpyruvate Carboxylase Isoforms in the Single-Cell, Facultative C4 System of Hydrilla verticillata1

    PubMed Central

    Rao, Srinath K.; Magnin, Noël C.; Reiskind, Julia B.; Bowes, George

    2002-01-01

    The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C4 plant. It typically exhibits C3 photosynthetic characteristics, but exposure to low [CO2] induces a C4 system in which the C4 and Calvin cycles co-exist in the same cell and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99% identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4, was exclusively expressed in leaves during C4 induction. This and enzyme kinetic data were consistent with it being the C4 photosynthesis isoform. However, the C4 signature serine of terrestrial plant C4 isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C3 sequences, was present. Western analyses of C3 and C4 leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C3, non-graminaceous C4, and Crassulacean acid metabolism PEPCs but not with the graminaceous C4, and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of other C4 angiosperms. PMID:12376652

  3. The source and characteristics of chemiluminescence associated with the oxygenase reaction catalyzed by Mn(2+)-ribulosebisphosphate carboxylase.

    PubMed

    Lilley, R M; Riesen, H; Andrews, T J

    1993-07-05

    We confirm the observation of Mogel and McFadden (Mogel, S.N., and McFadden, B. A. (1990) Biochemistry 29, 8333-8337) that ribulosebisphosphate carboxylase/oxygenase (rubisco) exhibits chemiluminescence while catalyzing its oxygenase reaction in the presence of Mn2+. However, our results with the spinach and Rhodospirillum rubrum enzymes differ markedly in the following respects. 1) Chemiluminescence intensity was directly proportional to enzyme concentration and behaved as if representing the rate of oxygenase catalysis. 2) The wavelength spectrum peaked at about 770 nm and extended beyond 810 nm. This seems inconsistent with chemiluminescence generated by simultaneous decay of pairs of singlet O2 molecules. It is consistent with manganese(II) luminescence and we discuss its possible sources. The time course of chemiluminescence (resolution, 0.25 s) was distinctively different for spinach and R. rubrum enzymes during the initial 5 s of catalysis, with the bacterial enzyme exhibiting a pronounced initial "burst." Chemiluminescence by the spinach enzyme responded to substrate concentrations in a manner consistent with known oxygenase properties, exhibiting Michaelis-Menten kinetics with ribulose-1,5-bisphosphate (Km 400 nM). Chemiluminescence required carbamylated enzyme with Mn2+ bound at the active site (activation energy, -57.1 KJ.mol-1). As an indicator of oxygenase activity, chemiluminescence represents an improvement over oxygen electrode measurements in response time and sensitivity by factors of at least 100.

  4. Mechanism of metamifop inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase in Echinochloa crus-galli

    PubMed Central

    Xia, Xiangdong; Tang, Wenjie; He, Shun; Kang, Jing; Ma, Hongju; Li, Jianhong

    2016-01-01

    Acetyl-coenzyme A carboxylase (ACCase) plays crucial roles in fatty acid metabolism and is an attractive target for herbicide discovery. Metamifop is a novel ACCase-inhibiting herbicide that can be applied to control sensitive weeds in paddy fields. In this study, the effects of metamifop on the chloroplasts, ACCase activity and carboxyltransferase (CT) domain gene expression in Echinochloa crus-galli were investigated. The results showed that metamifop interacted with the CT domain of ACCase in E. crus-galli. The three-dimensional structure of the CT domain of E. crus-galli ACCase in complex with metamifop was examined by homology modelling, molecular docking and molecular dynamics (MD) simulations. Metamifop has a different mechanism of inhibiting the CT domain compared with other ACCase inhibitors as it interacted with a different region in the active site of the CT domain. The protonation of nitrogen in the oxazole ring of metamifop plays a crucial role in the interaction between metamifop and the CT domain. The binding mode of metamifop provides a foundation for elucidating the molecular mechanism of target resistance and cross-resistance among ACCase herbicides, and for designing and optimizing ACCase inhibitors. PMID:27666674

  5. DNA inhibits catalysis by the carboxyltransferase subunit of acetyl-CoA carboxylase: implications for active site communication.

    PubMed

    Benson, Brian K; Meades, Glen; Grove, Anne; Waldrop, Grover L

    2008-01-01

    Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in the synthesis of long-chain fatty acids. The crystal structure of the Escherichia coli carboxyltransferase component of ACC revealed an alpha(2)beta(2) subunit composition with two active sites and, most importantly, a unique zinc domain in each alphabeta pair that is absent in the eukaryotic enzyme. We show here that carboxyltransferase binds DNA. Half-maximal saturation of different single-stranded or double-stranded DNA constructs is seen at 0.5-1.0 muM, and binding is cooperative and nonspecific. The substrates (malonyl-CoA and biocytin) inhibit DNA:carboxyltransferase complex formation. More significantly, single-stranded DNA, double-stranded DNA, and heparin inhibit the reaction catalyzed by carboxyltransferase, with single-stranded DNA and heparin acting as competitive inhibitors. However, double-inhibition experiments revealed that both DNA and heparin can bind the enzyme in the presence of a bisubstrate analog (BiSA), and the binding of BiSA has a very weak synergistic effect on the binding of the second inhibitor (DNA or heparin) and vice versa. In contrast, DNA and heparin can also bind to the enzyme simultaneously, but the binding of either molecule has a strong synergistic effect on binding of the other. An important mechanistic implication of these observations is that the dual active sites of ACC are functionally connected.

  6. Promotion of photosynthesis in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene by nitric oxide donors.

    PubMed

    Chen, Pingbo; Li, Xia; Huo, Kai; Wei, Xiaodong; Dai, Chuanchao; Lv, Chuangen

    2014-03-15

    We determined the effects of exogenous nitric oxide on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC). Seedlings were subjected to treatments with NO donors, an NO scavenger, phospholipase inhibitors, a Ca(2+) chelator, a Ca(2+) channel inhibitor, and a hydrogen peroxide (H2O2) inhibitor, individually and in various combinations. The NO donors significantly increased the net photosynthetic rate (PN) of PC and wild-type (WT), especially that of PC. Treatment with an NO scavenger did inhibit the PN of rice plants. The treatments with phospholipase inhibitors and a Ca(2+) chelator decreased the PN of WT and PC, and photosynthesis was more strongly inhibited in WT than in PC. Further analyses showed that the NO donors increased endogenous levels of NO and PLD activity, but decreased endogenous levels of Ca(2+) both WT and PC. However, there was a greater increase in NO in WT and a greater increase in PLD activity and Ca(2+) level in PC. The NO donors also increased both PEPC activity and pepc gene expression in PC. PEPC activity can be increased by SNP alone. But the expression of its encoding gene in PC might be regulated by SNP, together with PA and Ca(2+).

  7. Mechanism of metamifop inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase in Echinochloa crus-galli

    NASA Astrophysics Data System (ADS)

    Xia, Xiangdong; Tang, Wenjie; He, Shun; Kang, Jing; Ma, Hongju; Li, Jianhong

    2016-09-01

    Acetyl-coenzyme A carboxylase (ACCase) plays crucial roles in fatty acid metabolism and is an attractive target for herbicide discovery. Metamifop is a novel ACCase-inhibiting herbicide that can be applied to control sensitive weeds in paddy fields. In this study, the effects of metamifop on the chloroplasts, ACCase activity and carboxyltransferase (CT) domain gene expression in Echinochloa crus-galli were investigated. The results showed that metamifop interacted with the CT domain of ACCase in E. crus-galli. The three-dimensional structure of the CT domain of E. crus-galli ACCase in complex with metamifop was examined by homology modelling, molecular docking and molecular dynamics (MD) simulations. Metamifop has a different mechanism of inhibiting the CT domain compared with other ACCase inhibitors as it interacted with a different region in the active site of the CT domain. The protonation of nitrogen in the oxazole ring of metamifop plays a crucial role in the interaction between metamifop and the CT domain. The binding mode of metamifop provides a foundation for elucidating the molecular mechanism of target resistance and cross-resistance among ACCase herbicides, and for designing and optimizing ACCase inhibitors.

  8. A Different Mechanism for the Inhibition of the Carboxyltransferase Domain of Acetyl-coenzyme A Carboxylase by Tepraloxydim

    SciTech Connect

    Xiang, S.; Callaghan, M; Watson, K; Tong, L

    2009-01-01

    Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and are attractive targets for drug discovery. Haloxyfop and tepraloxydim belong to two distinct classes of commercial herbicides and kill sensitive plants by inhibiting the carboxyltransferase (CT) activity of ACC. Our earlier structural studies showed that haloxyfop is bound near the active site of the CT domain, at the interface of its dimer, and a large conformational change in the dimer interface is required for haloxyfop binding. We report here the crystal structure at 2.3 {angstrom} resolution of the CT domain of yeast ACC in complex with tepraloxydim. The compound has a different mechanism of inhibiting the CT activity compared to haloxyfop, as well as the mammalian ACC inhibitor CP-640186. Tepraloxydim probes a different region of the dimer interface and requires only small but important conformational changes in the enzyme, in contrast to haloxyfop. The binding mode of tepraloxydim explains the structure-activity relationship of these inhibitors, and provides a molecular basis for their distinct sensitivity to some of the resistance mutations, as compared to haloxyfop. Despite the chemical diversity between haloxyfop and tepraloxydim, the compounds do share two binding interactions to the enzyme, which may be important anchoring points for the development of ACC inhibitors

  9. Genome-wide Analysis of Phosphoenolpyruvate Carboxylase Gene Family and Their Response to Abiotic Stresses in Soybean

    PubMed Central

    Wang, Ning; Zhong, Xiujuan; Cong, Yahui; Wang, Tingting; Yang, Songnan; Li, Yan; Gai, Junyi

    2016-01-01

    Phosphoenolpyruvate carboxylase (PEPC) plays an important role in assimilating atmospheric CO2 during C4 and crassulacean acid metabolism photosynthesis, and also participates in various non-photosynthetic processes, including fruit ripening, stomatal opening, supporting carbon–nitrogen interactions, seed formation and germination, and regulation of plant tolerance to stresses. However, a comprehensive analysis of PEPC family in Glycine max has not been reported. Here, a total of ten PEPC genes were identified in soybean and denominated as GmPEPC1-GmPEPC10. Based on the phylogenetic analysis of the PEPC proteins from 13 higher plant species including soybean, PEPC family could be classified into two subfamilies, which was further supported by analyses of their conserved motifs and gene structures. Nineteen cis-regulatory elements related to phytohormones, abiotic and biotic stresses were identified in the promoter regions of GmPEPC genes, indicating their roles in soybean development and stress responses. GmPEPC genes were expressed in various soybean tissues and most of them responded to the exogenously applied phytohormones. GmPEPC6, GmPEPC8 and GmPEPC9 were significantly induced by aluminum toxicity, cold, osmotic and salt stresses. In addition, the enzyme activities of soybean PEPCs were also up-regulated by these treatments, suggesting their potential roles in soybean response to abiotic stresses. PMID:27924923

  10. ppc, the gene for phosphoenolpyruvate carboxylase from an extremely thermophilic bacterium, Rhodothermus obamensis: cloning, sequencing and overexpression in Escherichia coli.

    PubMed

    Takai, K; Sako, Y; Uchida, A

    1998-05-01

    The ppc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extremely thermophilic bacterium, Rhodothermus obamensis, was directly sequenced by the thermal asymmetric interlaced (TAIL) PCR method. An ORF for a 937 amino acid polypeptide was found in the gene. The ppc gene had a high G+C content (66.2 mol%) and the third position of the codon exhibited strong preference for G or C usage (85.0 mol%). The calculated molecular mass was 107,848 Da, which was consistent with the molecular mass of the enzyme as determined by SDS-PAGE (100 kDa). The amino acid sequence of R. obamensis PEPC was closely related to that of PEPC from another thermophile, a Thermus sp., and from a mesophile, Corynebacterium glutamicum, exhibiting 45.3% or 37.7% identity and 61.5% or 56.5% similarity, respectively. By Southern analysis, the ppc gene was found to be present in a single copy in the genomic DNA of this organism. The cloned gene was expressed in Escherichia coli using a pET expression vector system and a thermostable recombinant PEPC was obtained. Comparison of the deduced amino acid sequences of the thermophilic and mesophilic PEPCs revealed distinct or common preferences for specific amino acid composition and substitutions in the two thermophilic enzymes.

  11. Algal evolution in relation to atmospheric CO2: carboxylases, carbon-concentrating mechanisms and carbon oxidation cycles

    PubMed Central

    Raven, John A.; Giordano, Mario; Beardall, John; Maberly, Stephen C.

    2012-01-01

    Oxygenic photosynthesis evolved at least 2.4 Ga; all oxygenic organisms use the ribulose bisphosphate carboxylase-oxygenase (Rubisco)–photosynthetic carbon reduction cycle (PCRC) rather than one of the five other known pathways of autotrophic CO2 assimilation. The high CO2 and (initially) O2-free conditions permitted the use of a Rubisco with a high maximum specific reaction rate. As CO2 decreased and O2 increased, Rubisco oxygenase activity increased and 2-phosphoglycolate was produced, with the evolution of pathways recycling this inhibitory product to sugar phosphates. Changed atmospheric composition also selected for Rubiscos with higher CO2 affinity and CO2/O2 selectivity correlated with decreased CO2-saturated catalytic capacity and/or for CO2-concentrating mechanisms (CCMs). These changes increase the energy, nitrogen, phosphorus, iron, zinc and manganese cost of producing and operating Rubisco–PCRC, while biosphere oxygenation decreased the availability of nitrogen, phosphorus and iron. The majority of algae today have CCMs; the timing of their origins is unclear. If CCMs evolved in a low-CO2 episode followed by one or more lengthy high-CO2 episodes, CCM retention could involve a combination of environmental factors known to favour CCM retention in extant organisms that also occur in a warmer high-CO2 ocean. More investigations, including studies of genetic adaptation, are needed. PMID:22232762

  12. Genetic inhibition of hepatic acetyl-CoA carboxylase activity increases liver fat and alters global protein acetylationa

    PubMed Central

    Chow, Jenny D.Y.; Lawrence, Robert T.; Healy, Marin E.; Dominy, John E.; Liao, Jason A.; Breen, David S.; Byrne, Frances L.; Kenwood, Brandon M.; Lackner, Carolin; Okutsu, Saeko; Mas, Valeria R.; Caldwell, Stephen H.; Tomsig, Jose L.; Cooney, Gregory J.; Puigserver, Pere B.; Turner, Nigel; James, David E.; Villén, Judit; Hoehn, Kyle L.

    2014-01-01

    Lipid deposition in the liver is associated with metabolic disorders including fatty liver disease, type II diabetes, and hepatocellular cancer. The enzymes acetyl-CoA carboxylase 1 (ACC1) and ACC2 are powerful regulators of hepatic fat storage; therefore, their inhibition is expected to prevent the development of fatty liver. In this study we generated liver-specific ACC1 and ACC2 double knockout (LDKO) mice to determine how the loss of ACC activity affects liver fat metabolism and whole-body physiology. Characterization of LDKO mice revealed unexpected phenotypes of increased hepatic triglyceride and decreased fat oxidation. We also observed that chronic ACC inhibition led to hyper-acetylation of proteins in the extra-mitochondrial space. In sum, these data reveal the existence of a compensatory pathway that protects hepatic fat stores when ACC enzymes are inhibited. Furthermore, we identified an important role for ACC enzymes in the regulation of protein acetylation in the extra-mitochondrial space. PMID:24944901

  13. Interaction between potyvirus P3 and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of host plants.

    PubMed

    Lin, Lin; Luo, Zhaopeng; Yan, Fei; Lu, Yuwen; Zheng, Hongying; Chen, Jianping

    2011-08-01

    The P3 protein encoded by Shallot yellow stripe virus onion isolate (SYSV-O) interacted in the Yeast Two-hybrid (Y2H) system and in co-immunoprecipitation (Co-IP) assays with the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) protein that is encoded by the rbcL gene of its onion host. Dissection analysis by Y2H showed that the main part of SYSV P3 (amino acids 1-390) and onion RbcL (amino acids 1-137) were responsible for the interaction. The P3 proteins encoded by Onion yellow dwarf virus (OYDV), Soybean mosaic virus Pinellia isolate (SMV-P), and Turnip mosaic virus (TuMV) also interacted with RbcL, suggesting that a P3/RbcL interaction might exist generally for potyviruses. An interaction between P3 of these potyviruses and the small subunit of RubisCO (RbcS) was also demonstrated. Moreover, the P3N-PIPO protein encoded by a newly identified open reading frame embedded within the P3 cistron also interacted with both RbcL and RbcS. It is possible that the potyvirus P3 protein affects the normal functions of RubisCO which thus contributes to symptom development.

  14. Multiple isoforms of phosphoenolpyruvate carboxylase in the Orchidaceae (subtribe Oncidiinae): implications for the evolution of crassulacean acid metabolism

    PubMed Central

    Silvera, Katia; Winter, Klaus; Rodriguez, B. Leticia; Albion, Rebecca L.; Cushman, John C.

    2014-01-01

    Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and subsequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of transcriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and photosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independent origins. PMID:24913627

  15. Ion channels, phosphorylation and mammalian sperm capacitation

    PubMed Central

    Visconti, Pablo E; Krapf, Dario; de la Vega-Beltrán, José Luis; Acevedo, Juan José; Darszon, Alberto

    2011-01-01

    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies. PMID:21540868

  16. Nucleoside phosphorylation by the mineral schreibersite

    NASA Astrophysics Data System (ADS)

    Gull, Maheen; Mojica, Mike A.; Fernández, Facundo M.; Gaul, David A.; Orlando, Thomas M.; Liotta, Charles L.; Pasek, Matthew A.

    2015-11-01

    Phosphorylation of the nucleosides adenosine and uridine by the simple mixing and mild heating of aqueous solutions of the organic compounds with synthetic analogs of the meteoritic mineral schreibersite, (Fe,Ni)3P under slightly basic conditions (pH ~9) is reported. These results suggest a potential role for meteoritic phosphorus in the origin and development of early life.

  17. Phosphorylation of plastoglobular proteins in Arabidopsis thaliana

    PubMed Central

    Lohscheider, Jens N.; Friso, Giulia; van Wijk, Klaas J.

    2016-01-01

    Plastoglobules (PGs) are plastid lipid–protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles. PMID:26962209

  18. Phosphorylation of native porcine olfactory binding proteins.

    PubMed

    Nagnan-Le Meillour, Patricia; Le Danvic, Chrystelle; Brimau, Fanny; Chemineau, Philippe; Michalski, Jean-Claude

    2009-07-01

    The identification of various isoforms of olfactory binding proteins is of major importance to elucidate their involvement in detection of pheromones and other odors. Here, we report the characterization of the phosphorylation of OBP (odorant binding protein) and Von Ebner's gland protein (VEG) from the pig, Sus scrofa. After labeling with specific antibodies raised against the three types of phosphorylation (Ser, Thr, Tyr), the phosphate-modified residues were mapped by using the beta-elimination followed by Michael addition of dithiothreitol (BEMAD) method. Eleven phosphorylation sites were localized in the pOBP sequence and nine sites in the VEG sequence. OBPs are secreted by Bowman's gland cells in the extracellular mucus lining the nasal cavity. After tracking the secretion pathway in the rough endoplasmic reticulum of these cells, we hypothesize that these proteins may be phosphorylated by ectokinases that remain to be characterized. The existence of such a regulatory mechanism theoretically increases the number of OBP variants, and it suggests a more specific role for OBPs in odorant coding than the one of odorant solubilizer and transporter.

  19. Function of Estrogen Receptor Tryosine Phosphorylation

    DTIC Science & Technology

    1997-07-01

    localization of the receptors, ligand binding, DNA binding, transcriptional activation, and receptor turnover ( LeGoff et al. 1994; Lahooti et al. 1994...1040-1049 (1995). LeGoff P., M.M. Montano, D.J. Schodin, and B. Katzenellenbogen. Phosphorylation of the Human Estrogen Receptor. J. Biol. Chem

  20. Phosphoryl Transfer Reaction Snapshots in Crystals

    PubMed Central

    Gerlits, Oksana; Tian, Jianhui; Das, Amit; Langan, Paul; Heller, William T.; Kovalevsky, Andrey

    2015-01-01

    To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca2+ cations with Mg2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. The present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date. PMID:25925954

  1. Pathogenic PS1 phosphorylation at Ser367

    PubMed Central

    Maesako, Masato; Horlacher, Jana; Zoltowska, Katarzyna M; Kastanenka, Ksenia V; Kara, Eleanna; Svirsky, Sarah; Keller, Laura J; Li, Xuejing; Hyman, Bradley T; Bacskai, Brian J; Berezovska, Oksana

    2017-01-01

    The high levels of serine (S) and threonine (T) residues within the Presenilin 1 (PS1) N-terminus and in the large hydrophilic loop region suggest that the enzymatic function of PS1/γ-secretase can be modulated by its ‘phosphorylated’ and ‘dephosphorylated’ states. However, the functional outcome of PS1 phosphorylation and its significance for Alzheimer’s disease (AD) pathogenesis is poorly understood. Here, comprehensive analysis using FRET-based imaging reveals that activity-driven and Protein Kinase A-mediated PS1 phosphorylation at three domains (domain 1: T74, domain 2: S310 and S313, domain 3: S365, S366, and S367), with S367 being critical, is responsible for the PS1 pathogenic ‘closed’ conformation, and resulting increase in the Aβ42/40 ratio. Moreover, we have established novel imaging assays for monitoring PS1 conformation in vivo, and report that PS1 phosphorylation induces the pathogenic conformational shift in the living mouse brain. These phosphorylation sites represent potential new targets for AD treatment. DOI: http://dx.doi.org/10.7554/eLife.19720.001 PMID:28132667

  2. Regulation of protein phosphorylation in oat mitochondria

    SciTech Connect

    Pike, C.; Kopeck, K.; Sceppa, E. )

    1989-04-01

    We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with {sup 32}P-{gamma}-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of {sup 35}S-adenosine thiotriphosphate.

  3. Controlling cytokinesis through promiscuous phosphorylation outside BARs.

    PubMed

    Glotzer, Michael

    2010-07-09

    In this issue of Molecular Cell, Roberts-Galbraith and colleagues report that a key cytokinetic regulator in fission yeast, Cdc15, is phosphorylated on numerous sites that collectively, but not individually, control its oligomerization state and its associations with the plasma membrane and interacting proteins.

  4. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  5. Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

    SciTech Connect

    Smiley, R.M. Columbia Univ College of Physicians and Surgeons, New York, NY ); Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J. )

    1990-01-01

    The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with {sup 32}PO{sub 4} in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the {beta}-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M{sub r} 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the {alpha}-2 agonist clonidine. Epinephrine, a combined {alpha} and {beta} agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the {alpha}-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.

  6. Changes in tau phosphorylation in hibernating rodents.

    PubMed

    León-Espinosa, Gonzalo; García, Esther; García-Escudero, Vega; Hernández, Félix; Defelipe, Javier; Avila, Jesús

    2013-07-01

    Tau is a cytoskeletal protein present mainly in the neurons of vertebrates. By comparing the sequence of tau molecule among different vertebrates, it was found that the variability of the N-terminal sequence in tau protein is higher than that of the C-terminal region. The N-terminal region is involved mainly in the binding of tau to cellular membranes, whereas the C-terminal region of the tau molecule contains the microtubule-binding sites. We have compared the sequence of Syrian hamster tau with the sequences of other hibernating and nonhibernating rodents and investigated how differences in the N-terminal region of tau could affect the phosphorylation level and tau binding to cell membranes. We also describe a change, in tau phosphorylation, on a casein kinase 1 (ck1)-dependent site that is found only in hibernating rodents. This ck1 site seems to play an important role in the regulation of tau binding to membranes.

  7. MAP kinases phosphorylate rice WRKY45.

    PubMed

    Ueno, Yoshihisa; Yoshida, Riichiro; Kishi-Kaboshi, Mitsuko; Matsushita, Akane; Jiang, Chang-Jie; Goto, Shingo; Takahashi, Akira; Hirochika, Hirohiko; Takatsuji, Hiroshi

    2013-06-01

    WRKY45 transcription factor is a central regulator of disease resistance mediated by the salicylic acid (SA) signaling pathway in rice. SA-activated WRKY45 protein induces the accumulation of its own mRNA. However, the mechanism underlying this regulation is still unknown. Here, we report three lines of evidence showing that a mitogen-activated protein kinase (MAPK) cascade is involved in this regulation. An inhibitor of MAPK kinase (MAPKK) suppressed the increase in WRKY45 transcript level in response to SA. Two MAPKs, OsMPK4 and OsMPK6, phosphorylated WRKY45 protein in vitro. The activity of OsMPK6 was rapidly upregulated by SA treatment in rice cells. These results suggest that WRKY45 is regulated by MAPK-dependent phosphorylation in the SA pathway.

  8. Phosphorylation of Kraft fibers with phosphate esters.

    PubMed

    Shi, Ying; Belosinschi, Dan; Brouillette, François; Belfkira, Ahmed; Chabot, Bruno

    2014-06-15

    Phosphate esters, derived from two different long-chain aliphatic alcohols, were used as phosphorylating reagents for Kraft pulp fibers. High phosphorus contents and almost non-degraded fibers were obtained by following this pathway. The phosphorylation efficiency was influenced by the alkyl chain length of PEs since the phosphorus content in modified fibers was higher for the shorter chain reagent. Due to the heterogeneous reaction environment, the amount of grafted phosphorus was found to be almost three times higher at the surface than in the bulk of the fibers. Analyses also indicated that the phosphorus was bonded to fibers as a phosphate-like structure. Furthermore, the situation seemed to be different for the fiber surface where significant amounts of phosphorus were present in more complex structures like pyrophosphate or even oligo-phosphate.

  9. Drosophila melanogaster Acetyl-CoA-carboxylase sustains a fatty acid-dependent remote signal to waterproof the respiratory system.

    PubMed

    Parvy, Jean-Philippe; Napal, Laura; Rubin, Thomas; Poidevin, Mickael; Perrin, Laurent; Wicker-Thomas, Claude; Montagne, Jacques

    2012-01-01

    Fatty acid (FA) metabolism plays a central role in body homeostasis and related diseases. Thus, FA metabolic enzymes are attractive targets for drug therapy. Mouse studies on Acetyl-coenzymeA-carboxylase (ACC), the rate-limiting enzyme for FA synthesis, have highlighted its homeostatic role in liver and adipose tissue. We took advantage of the powerful genetics of Drosophila melanogaster to investigate the role of the unique Drosophila ACC homologue in the fat body and the oenocytes. The fat body accomplishes hepatic and storage functions, whereas the oenocytes are proposed to produce the cuticular lipids and to contribute to the hepatic function. RNA-interfering disruption of ACC in the fat body does not affect viability but does result in a dramatic reduction in triglyceride storage and a concurrent increase in glycogen accumulation. These metabolic perturbations further highlight the role of triglyceride and glycogen storage in controlling circulatory sugar levels, thereby validating Drosophila as a relevant model to explore the tissue-specific function of FA metabolic enzymes. In contrast, ACC disruption in the oenocytes through RNA-interference or tissue-targeted mutation induces lethality, as does oenocyte ablation. Surprisingly, this lethality is associated with a failure in the watertightness of the spiracles-the organs controlling the entry of air into the trachea. At the cellular level, we have observed that, in defective spiracles, lipids fail to transfer from the spiracular gland to the point of air entry. This phenotype is caused by disrupted synthesis of a putative very-long-chain-FA (VLCFA) within the oenocytes, which ultimately results in a lethal anoxic issue. Preventing liquid entry into respiratory systems is a universal issue for air-breathing animals. Here, we have shown that, in Drosophila, this process is controlled by a putative VLCFA produced within the oenocytes.

  10. Purification and characterization of large and small subunits of ribulose 1,5-bisphosphate carboxylase expressed separately in Escherichia coli.

    PubMed

    Smrcka, A V; Ramage, R T; Bohnert, H J; Jensen, R G

    1991-04-01

    Procedures were developed for 95 and 80% purification to homogeneity of the large subunit (L) and small subunit (S) of ribulose 1,5-bisphosphate carboxylase/oxygenase (L8S8) from Synechococcus PCC 6301, each expressed separately in Escherichia coli. Purified L had a low specific activity in the absence of S (0.075 mumol CO2 fixed/mg holoenzyme/min). Following elution on a Pharmacia Superose 6 or 12 gel filtration column, 50% of the purified L appeared as the octamer, L8. The rest was in equilibrium with lower polymeric species and/or was retained on the column. Large and small subunits assembled rapidly into the L8S8 holoenzyme that had high specific activities, 6.2 and 3.1 mumol CO2 fixed/mg holoenzyme/min for the homologous Synechococcus L8S8 and the hybrid Synechococcus L-pea S L8S8, respectively. The CO2 dependence for carbamylation of L8 was compared to that of L8S8 as a function of pH and CO2 concentration. The pH dependence indicated an apparent pKa for L8 of 8.28 and for L8S8 of 8.15, suggesting that S may influence the pKa of the lysine involved in carbamylation. The Kact for CO2 at pH 8.4 were similar for L8 (13.5 microM) and L8S8 (15.5 microM). L8 bound 2-[14C]carboxy-D-arabinitol 1,5-bisphosphate (CABP) tightly so that most of the bound [14C]CABP survived gel filtration. A major amount of the L8-[14C]CABP complex appeared as larger polymeric aggregates when eluted in the presence of E. coli protein.

  11. Characterization of the duplicate ribulose-1,5-bisphosphate carboxylase genes and cbb promoters of Alcaligenes eutrophus.

    PubMed

    Kusian, B; Bednarski, R; Husemann, M; Bowien, B

    1995-08-01

    Autotrophic CO2 fixation via the Calvin carbon reduction cycle in Alcaligenes eutrophus H16 is genetically determined by two highly homologous cbb operons, one of which is located on the chromosome and the other on megaplasmid pHG1 of the organism. An activator gene, cbbR, lies in divergent orientation only 167 bp upstream of the chromosomal operon and controls the expression of both cbb operons. The two 5'-terminal genes of the operons, cbbLS, coding for ribulose-1,5-bisphosphate carboxylase/oxygenase, were sequenced. Mapping of the 5' termini of the 2.1-kb cbbLS transcripts by primer extension and by nuclease S1 treatment revealed a single transcriptional start point at the same relative position for the chromosomal and plasmid-borne cbb operons. The derived cbb operon promoter showed similarity to sigma 70-dependent promoters of Escherichia coli. For the 1.4-kb transcripts of cbbR, the transcriptional start points were different in autotrophic and heterotrophic cells. The two corresponding cbbR promoters overlapped the cbb operon promoter and also displayed similarities to sigma 70-dependent promoters. The deficient cbbR gene located on pHG1 was transcribed as well. A newly constructed double operon fusion vector was used to determine the activities of the cbb promoters. Fusions with fragments carrying the cbb intergenic control regions demonstrated that the cbb operon promoters were strongly regulated in response to autotrophic versus heterotrophic growth conditions. In contrast, the cbbR promoters displayed low constitutive activities. The data suggest that the chromosomal and plasmid-borne cbb promoters of A. eutrophus H16 are functionally equivalent despite minor structural differences.

  12. A Chemogenomic Screen Reveals Novel Snf1p/AMPK Independent Regulators of Acetyl-CoA Carboxylase.

    PubMed

    Bozaquel-Morais, Bruno L; Madeira, Juliana B; Venâncio, Thiago M; Pacheco-Rosa, Thiago; Masuda, Claudio A; Montero-Lomeli, Monica

    2017-01-01

    Acetyl-CoA carboxylase (Acc1p) is a key enzyme in fatty acid biosynthesis and is essential for cell viability. To discover new regulators of its activity, we screened a Saccharomyces cerevisiae deletion library for increased sensitivity to soraphen A, a potent Acc1p inhibitor. The hits identified in the screen (118 hits) were filtered using a chemical-phenotype map to exclude those associated with pleiotropic drug resistance. This enabled the identification of 82 ORFs that are genetic interactors of Acc1p. The main functional clusters represented by these hits were "transcriptional regulation", "protein post-translational modifications" and "lipid metabolism". Further investigation of the "transcriptional regulation" cluster revealed that soraphen A sensitivity is poorly correlated with ACC1 transcript levels. We also studied the three top unknown ORFs that affected soraphen A sensitivity: SOR1 (YDL129W), SOR2 (YIL092W) and SOR3 (YJR039W). Since the C18/C16 ratio of lipid acyl lengths reflects Acc1p activity levels, we evaluated this ratio in the three mutants. Deletion of SOR2 and SOR3 led to reduced acyl lengths, suggesting that Acc1p is indeed down-regulated in these strains. Also, these mutants showed no differences in Snf1p/AMPK activation status and deletion of SNF1 in these backgrounds did not revert soraphen A sensitivity completely. Furthermore, plasmid maintenance was reduced in sor2Δ strain and this trait was shared with 18 other soraphen A sensitive hits. In summary, our screen uncovered novel Acc1p Snf1p/AMPK-independent regulators.

  13. Covalent modification of a highly reactive and essential lysine residue of ribulose-1,5-bisphosphate carboxylase/oxygenase activase.

    PubMed

    Salvucci, M E

    1993-10-01

    Chemical modification of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters was used to identify a reactive lysyl residue that is essential for activity. Incubation of Rubisco activase with sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS) or sulfosuccinimidyl-acetate (sulfo-NHS-acetate) caused progressive inactivation of ATPase activity and concomitant loss of the ability to activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco activase, exhibiting a second-order rate constant for inactivation of 239 M-1 s-1 compared to 21 M-1 s-1 for sulfo-NHS-acetate. Inactivation of enzyme activity by AMCA-sulfo-NHS correlated with the incorporation of 1.9 mol of AMCA per mol of 42-kD Rubisco activase monomer. ADP, a competitive inhibitor of Rubisco activase, afforded considerable protection against inactivation of Rubisco activase and decreased the amount of AMCA incorporated into the Rubisco activase monomer. Sequence analysis of the major labeled peptide from AMCA-sulfo-NHS-modified enzyme showed that the primary site of modification was lysine-247 (K247) in the tetrapeptide methionine-glutamic acid-lysine-phenylalanine. Upon complete inactivation of ATPase activity, modification of K247 accounted for 1 mol of AMCA incorporated per mol of Rubisco activase monomer. Photoaffinity labeling of AMCA-sulfo-NHS- and sulfo-NHS-acetate-modified Rubisco activase with ATP analogs derivatized on either the adenine base or on the gamma-phosphate showed that K247 is not essential for the binding of adenine nucleotides per se. Instead, the data indicated that the essentiality of K247 is probably due to an involvement of this highly reactive, species-invariant residue in an obligatory interaction that occurs between the protein and the nucleotide phosphate during catalysis.

  14. Changes in the net charge and subunit properties of ribulose bisphosphate carboxylase--oxygenase during cold hardening of Puma rye.

    PubMed

    Huner, N P; Macdowall, F D

    1979-02-01

    Ribulose bisphosphate carboxylase--oxygenase (RUBPCase) from leaves of cold-hardened and unhardened Puma rye was purified by gel filtration and ion exchange chromatography. The specific activity of the hardened form was twice that of the unhardened form. A difference in charge between the two forms of this enzyme was proved by gel electrofocussing. The estimated isoelectric point (pI) values were 6.4 and 6.3 for the enzyme from the hardened and unhardened source respectively. The large subunit (55,000 molecular weight) of the enzyme from only the unhardened source formed at apparent dimer during sodium dodecyl sulfate (SDS) gel electrophoresis. At pH 6,8 it was also the source of an anomalous polypeptide with an apparent molecular weight of 47,000. This anomalous polypeptide appeared in both hardened and unhardened preparations after irreversible inactivation of RUBPCase activity by NaCl. It also appeared after preparation of the purified enzymes for SDS--PAGE in the absence of beta-mercaptoethanol, but this was reversible. The enzyme from the hardened source was less affected in the absence of reducing agent. Structural evidence was obtained for the previously reported cold hardening of the enzyme against freeze inactivation. A freeze-thaw cycle applied to the enzyme in vitro caused some polymerization of the large subunit and its anomalous polypeptide, in the absence of reducing agent, especially in the unhardened case. This increased with repeated cycles until the fifth cycle when the large subunit monomer and its satellite were abolished only in preparations from the unhardened source. These data indicate that the large subunit is a probable site of change that occurred in this enzyme during cold hardening.

  15. Altered intersubunit interactions in crystal structures of catalytically compromised ribulose-1,5-bisphosphate carboxylase/oxygenase.

    PubMed

    Karkehabadi, Saeid; Taylor, Thomas C; Spreitzer, Robert J; Andersson, Inger

    2005-01-11

    Substitution of Leu290 by Phe (L290F) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from the unicellular green alga Chlamydomonas reinhardtii causes a 13% decrease in CO(2)/O(2) specificity and reduced thermal stability. Genetic selection for restored photosynthesis at the restrictive temperature identified an Ala222 to Thr (A222T) substitution that suppresses the deleterious effects of the original mutant substitution to produce a revertant enzyme with improved thermal stability and kinetic properties virtually indistinguishable from that of the wild-type enzyme. Because the mutated residues are situated approximately 19 A away from the active site, they must affect the relative rates of carboxylation and oxygenation in an indirect way. As a means for elucidating the role of such distant interactions in Rubisco catalysis and stability, we have determined the crystal structures of the L290F mutant and L290F/A222T revertant enzymes to 2.30 and 2.05 A resolution, respectively. Inspection of the structures reveals that the mutant residues interact via van der Waals contacts within the same large subunit (intrasubunit path, 15.2 A Calpha-Calpha) and also via a path involving a neighboring small subunit (intersubunit path, 18.7 A Calpha-Calpha). Structural analysis of the mutant enzymes identified regions (residues 50-72 of the small subunit and residues 161-164 and 259-264 of the large subunit) that show significant and systematically increased atomic temperature factors in the L290F mutant enzyme compared to wild type. These regions coincide with residues on the interaction paths between the L290F mutant and A222T suppressor sites and could explain the temperature-conditional phenotype of the L290F mutant strain. This suggests that alterations in subunit interactions will influence protein dynamics and, thereby, affect catalysis.

  16. Presence of a structurally novel type ribulose-bisphosphate carboxylase/oxygenase in the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1.

    PubMed

    Ezaki, S; Maeda, N; Kishimoto, T; Atomi, H; Imanaka, T

    1999-02-19

    We have characterized the gene encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoded a protein consisting of 444 amino acid residues, corresponding in size to the large subunit of previously reported Rubiscos. Rubisco of P. kodakaraensis KOD1 (Pk-Rubisco) showed only 51.4% similarity with the large subunit of type I Rubisco from spinach and 47.3% with that of type II Rubisco from Rhodospirillum rubrum, suggesting that the enzyme was not a member of either type. Active site residues identified from type I and type II Rubiscos were conserved. We expressed the gene in Escherichia coli, and we obtained a soluble protein with the expected molecular mass and N-terminal amino acid sequence. Purification of the recombinant protein revealed that Pk-Rubisco was an L8 type homo-octamer. Pk-Rubisco showed highest specific activity of 19.8 x 10(3) nmol of CO2 fixed per min/mg, and a tau value of 310 at 90 degreesC, both higher than any previously characterized Rubisco. The optimum pH was 8.3, and the enzyme possessed extreme thermostability, with a half-life of 15 h at 80 degreesC. Northern blot analysis demonstrated that the gene was transcribed in P. kodakaraensis KOD1. Furthermore, Western blot analysis with cell-free extract of P. kodakaraensis KOD1 clearly indicated the presence of Pk-Rubisco in the native host cells.

  17. Ribulose-1,5-bisphosphate carboxylase/oxygenase gene expression and diversity of Lake Erie planktonic microorganisms

    SciTech Connect

    Xu, H.H.; Tabita, F.R.

    1996-06-01

    Carbon dioxide fixation is carried out primarily through the Calvin-Benson-Bassham reductive pentose phosphate cycle, in which rubulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme. The primary structure of the large subunit of form I RubisCO is well conserved; however, four distinct types, A, B, C, and D, may be distinguished. To better understand the environmental regulation of RubisCO in Lake Erie phytoplanktonic microorganisms, we have isolated total RNA and DNA from four Lake Erie sampling sites. Probes prepared from RubisCO large-subunit genes (rbcL) of the freshwater cyanobacterium Synechococcus sp. strain PCC6301 (representative of type IB) and the diatom Cylindrotheca sp. strain N1 (representative of type ID) was determined. It appeared that type ID (diatom) rbcL gene expression per gene dose decreased as the sampling sites shifted toward open water. By contrast, a similar trend was not observed for cyanobacterial (type IB) rbcL gene expression per gene dose. Thus far, a total of 21 clones of rbcL genes derived from mRNA have been obtained and completely sequenced from the Ballast Island site. For surface water samples, deduced amino acid sequences of five of six clones appeared to be representative of green algae. In contrast, six of nine sequenced rbcL clones from 10-m-deep samples were a chromophytic and rhodophytic lineages. At 5 m deep, the active CO{sub 2}-fixing planktonic organisms represented a diverse group, including organisms related to Chlorella ellipsoidea, Cylindrotheca sp. strain N1, and Olisthodiscus luteus. Although many more samplings at diverse sites must be accomplished, the discovery of distinctly different sequences of rbcL mRNA at different water depths suggests that there is a stratification of active CO{sub 2}-fixing organisms in western Lake Erie. 54 refs., 7 figs.

  18. Graminicide insensitivity correlates with herbicide-binding co-operativity on acetyl-CoA carboxylase isoforms.

    PubMed

    Price, Lindsey J; Herbert, Derek; Moss, Stephen R; Cole, David J; Harwood, John L

    2003-10-15

    The sensitivity of grass species to important classes of graminicide herbicides inhibiting ACCase (acetyl-CoA carboxylase) is associated with a specific inhibition of the multifunctional ACCase located in the plastids of grasses. In contrast, the multisubunit form of ACCase found in the chloroplasts of dicotyledonous plants is insensitive and the minor cytosolic multifunctional isoforms of the enzyme in both types of plants are also less sensitive to inhibition. We have isolated, separated and characterized the multifunctional ACCase isoforms found in exceptional examples of grasses that are either inherently insensitive to these graminicides, or from biotypes showing acquired resistance to their use. Major and minor multifunctional enzymes were isolated from cell suspension cultures of Festuca rubra and the 'Notts A1'-resistant biotype of Alopecurus myosuroides, and their properties compared with those isolated from cells of wild-type sensitive A. myosuroides or from sensitive maize. Purifications of up to 300-fold were necessary to separate the two isoforms. The molecular masses (200-230 kDa) and K(m) values for all three substrates (ATP, bicarbonate and acetyl-CoA) were similar for the different ACCases, irrespective of their graminicide sensitivity. Moreover, we found no correlation between the ability of isoforms to carboxylate propionyl-CoA and their sensitivity to graminicides. However, insensitive purified forms of ACCase were characterized by herbicide-binding co-operativity, whereas, in contrast, sensitive forms of the enzymes were not. Our studies on isolated individual isoforms of ACCase from grasses support and extend previous indications that herbicide binding co-operativity is the only kinetic property that differentiates naturally or selected insensitive enzymes from the typical sensitive forms usually found in grasses.

  19. A Chemogenomic Screen Reveals Novel Snf1p/AMPK Independent Regulators of Acetyl-CoA Carboxylase

    PubMed Central

    Bozaquel-Morais, Bruno L.; Madeira, Juliana B.; Venâncio, Thiago M.; Pacheco-Rosa, Thiago; Masuda, Claudio A.; Montero-Lomeli, Monica

    2017-01-01

    Acetyl-CoA carboxylase (Acc1p) is a key enzyme in fatty acid biosynthesis and is essential for cell viability. To discover new regulators of its activity, we screened a Saccharomyces cerevisiae deletion library for increased sensitivity to soraphen A, a potent Acc1p inhibitor. The hits identified in the screen (118 hits) were filtered using a chemical-phenotype map to exclude those associated with pleiotropic drug resistance. This enabled the identification of 82 ORFs that are genetic interactors of Acc1p. The main functional clusters represented by these hits were “transcriptional regulation”, “protein post-translational modifications” and “lipid metabolism”. Further investigation of the “transcriptional regulation” cluster revealed that soraphen A sensitivity is poorly correlated with ACC1 transcript levels. We also studied the three top unknown ORFs that affected soraphen A sensitivity: SOR1 (YDL129W), SOR2 (YIL092W) and SOR3 (YJR039W). Since the C18/C16 ratio of lipid acyl lengths reflects Acc1p activity levels, we evaluated this ratio in the three mutants. Deletion of SOR2 and SOR3 led to reduced acyl lengths, suggesting that Acc1p is indeed down-regulated in these strains. Also, these mutants showed no differences in Snf1p/AMPK activation status and deletion of SNF1 in these backgrounds did not revert soraphen A sensitivity completely. Furthermore, plasmid maintenance was reduced in sor2Δ strain and this trait was shared with 18 other soraphen A sensitive hits. In summary, our screen uncovered novel Acc1p Snf1p/AMPK-independent regulators. PMID:28076367

  20. Positive selection of Kranz and non-Kranz C4 phosphoenolpyruvate carboxylase amino acids in Suaedoideae (Chenopodiaceae).

    PubMed

    Rosnow, Josh J; Edwards, Gerald E; Roalson, Eric H

    2014-07-01

    In subfamily Suaedoideae, four independent gains of C4 photosynthesis are proposed, which includes two parallel origins of Kranz anatomy (sections Salsina and Schoberia) and two independent origins of single-cell C4 anatomy (Bienertia and Suaeda aralocaspica). Additional phylogenetic support for this hypothesis was generated from sequence data of the C-terminal portion of the phosphoenolpyruvate carboxylase (PEPC) gene used in C4 photosynthesis (ppc-1) in combination with previous sequence data. ppc-1 sequence was generated for 20 species in Suaedoideae and two outgroup Salsola species that included all types of C4 anatomies as well as two types of C3 anatomies. A branch-site test for positively selected codons was performed using the software package PAML. From labelling of the four branches where C4 is hypothesized to have developed (foreground branches), residue 733 (maize numbering) was identified to be under positive selection with a posterior probability >0.99 and residue 868 at the >0.95 interval using Bayes empirical Bayes (BEB). When labelling all the branches within C4 clades, the branch-site test identified 13 codons to be under selection with a posterior probability >0.95 by BEB; this is discussed considering current information on functional residues. The signature C4 substitution of an alanine for a serine at position 780 in the C-terminal end (which is considered a major determinant of affinity for PEP) was only found in four of the C4 species sampled, while eight of the C4 species and all the C3 species have an alanine residue; indicating that this substitution is not a requirement for C4 function.

  1. Coordinate, Organ-Specific and Developmental Regulation of Ribulose 1,5-Bisphosphate Carboxylase Gene Expression in Amaranthus hypochondriacus1

    PubMed Central

    Nikolau, Basil J.; Klessig, Daniel F.

    1987-01-01

    The expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in roots, stems, cotyledons, and leaves of amaranth during the development of these tissues. The highest accumulation of LSU and SSU polypeptides occurred in cotyledons and leaves. Their steady state levels were approximately 20-fold lower in stems, while in roots neither LSU and SSU polypeptides nor their respective mRNAs could be detected. In cotyledons and leaves accumulation of these two polypeptides reached peak levels during the expansion stage of each tissue and then declined, reflecting changes in the synthesis, not turnover, of these proteins. In cotyledons and stems, the rates of synthesis of LSU and SSU polypeptides correlated with the levels of their respective mRNA, suggesting regulation primarily at the transcriptional level. In contrast, the dramatic and specific decrease in the synthesis of these two proteins during the last stages of development of the leaves could only partially be accounted for by the modest reduction in their mRNAs. Neither the translatability of these mRNAs, as assayed in cell-free systems, nor the stability of LSU and SSU polypeptides were altered, thus implying that control was being exerted at the translational level. During the development of these different organs, the expression of the LSU and SSU genes were generally coordinately regulated both at the levels of protein synthesis and mRNA accumulation. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16665651

  2. Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

    PubMed Central

    Paul, K; Morell, M K; Andrews, T J

    1993-01-01

    The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme. PMID:8278544

  3. Characterization of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase activase isoforms reveals hexameric assemblies with increased thermal stability.

    PubMed

    Keown, Jeremy R; Pearce, Frederick Grant

    2014-12-15

    Most plants contain two isoforms of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a chloroplast protein that maintains the activity of Rubisco during photosynthesis. The longer (α-) Rca isoform has previously been shown to regulate the activity of Rubisco in response to both the ADP:ATP ratio and redox potential via thioredoxin-f. We have characterized the arrangement of the different spinach (Spinacia oleracea) isoforms in solution, and show how the presence of nucleotides changes the oligomeric state. Although the shorter (β-) isoform from both tobacco (Nicotiana tabacum) and spinach tend to form a range of oligomers in solution, the size of which are relatively unaffected by the addition of nucleotide, the spinach α-isoform assembles as a hexamer in the presence of adenosine 5'-[γ-thio]triphosphate (ATPγS). These hexamers have significantly higher heat stability, and may play a role in optimizing photosynthesis at higher temperatures. Hexamers were also observed for mixtures of the two isoforms, suggesting that the α-isoform can act as a structural scaffold for hexamer formation by the β-isoform. Additionally, it is shown that a variant of the tobacco β-isoform acts in a similar fashion to the α-isoform of spinach, forming thermally stable hexamers in the presence of ATPγS. Both isoforms had similar rates of ATP hydrolysis, suggesting that a propensity for hexamer formation may not necessarily be correlated with activity. Modelling of the hexameric structures suggests that although the N-terminus of Rca forms a highly dynamic, extended structure, the C-terminus is located adjacent to the intersubunit interface.

  4. Solid polymer electrolyte from phosphorylated chitosan

    SciTech Connect

    Fauzi, Iqbal Arcana, I Made

    2014-03-24

    Recently, the need of secondary battery application continues to increase. The secondary battery which using a liquid electrolyte was indicated had some weakness. A solid polymer electrolyte is an alternative electrolytes membrane which developed in order to replace the liquid electrolyte type. In the present study, the effect of phosphorylation on to polymer electrolyte membrane which synthesized from chitosan and lithium perchlorate salts was investigated. The effect of the component’s composition respectively on the properties of polymer electrolyte, was carried out by analyzed of it’s characterization such as functional groups, ion conductivity, and thermal properties. The mechanical properties i.e tensile resistance and the morphology structure of membrane surface were determined. The phosphorylation processing of polymer electrolyte membrane of chitosan and lithium perchlorate was conducted by immersing with phosphoric acid for 2 hours, and then irradiated on a microwave for 60 seconds. The degree of deacetylation of chitosan derived from shrimp shells was obtained around 75.4%. Relative molecular mass of chitosan was obtained by viscometry method is 796,792 g/mol. The ionic conductivity of chitosan membrane was increase from 6.33 × 10{sup −6} S/cm up to 6.01 × 10{sup −4} S/cm after adding by 15 % solution of lithium perchlorate. After phosphorylation, the ionic conductivity of phosphorylated lithium chitosan membrane was observed 1.37 × 10{sup −3} S/cm, while the tensile resistance of 40.2 MPa with a better thermal resistance. On the strength of electrolyte membrane properties, this polymer electrolyte membrane was suggested had one potential used for polymer electrolyte in field of lithium battery applications.

  5. Mixed mechanisms of multi-site phosphorylation

    PubMed Central

    Suwanmajo, Thapanar; Krishnan, J.

    2015-01-01

    Multi-site phosphorylation is ubiquitous in cell biology and has been widely studied experimentally and theoretically. The underlying chemical modification mechanisms are typically assumed to be distributive or processive. In this paper, we study the behaviour of mixed mechanisms that can arise either because phosphorylation and dephosphorylation involve different mechanisms or because phosphorylation and/or dephosphorylation can occur through a combination of mechanisms. We examine a hierarchy of models to assess chemical information processing through different mixed mechanisms, using simulations, bifurcation analysis and analytical work. We demonstrate how mixed mechanisms can show important and unintuitive differences from pure distributive and processive mechanisms, in some cases resulting in monostable behaviour with simple dose–response behaviour, while in other cases generating new behaviour-like oscillations. Our results also suggest patterns of information processing that are relevant as the number of modification sites increases. Overall, our work creates a framework to examine information processing arising from complexities of multi-site modification mechanisms and their impact on signal transduction. PMID:25972433

  6. Mixed mechanisms of multi-site phosphorylation.

    PubMed

    Suwanmajo, Thapanar; Krishnan, J

    2015-06-06

    Multi-site phosphorylation is ubiquitous in cell biology and has been widely studied experimentally and theoretically. The underlying chemical modification mechanisms are typically assumed to be distributive or processive. In this paper, we study the behaviour of mixed mechanisms that can arise either because phosphorylation and dephosphorylation involve different mechanisms or because phosphorylation and/or dephosphorylation can occur through a combination of mechanisms. We examine a hierarchy of models to assess chemical information processing through different mixed mechanisms, using simulations, bifurcation analysis and analytical work. We demonstrate how mixed mechanisms can show important and unintuitive differences from pure distributive and processive mechanisms, in some cases resulting in monostable behaviour with simple dose-response behaviour, while in other cases generating new behaviour-like oscillations. Our results also suggest patterns of information processing that are relevant as the number of modification sites increases. Overall, our work creates a framework to examine information processing arising from complexities of multi-site modification mechanisms and their impact on signal transduction.

  7. Function of platelet 47K protein phosphorylation

    SciTech Connect

    Imaoka, T.

    1987-05-01

    To provide insight into the biochemical pathway of platelet activation, they purified both unphosphorylated and phosphorylated P47 to homogeneity from human platelets. This study represents the first demonstration of a change of physiological action of P47 in response to phosphorylation in platelet activation. SVI labelled unphosphorylated P47 had an ability to bind with platelet membrane fraction in the presence of phosphatidylserine. Effect of diacylglycerol was inhibitory in this PS dependent P47 binding with membrane. Unphosphorylated P47 had an inhibitory activity in platelet actin polymerization. Molar ratio to inhibit actin polymerization was 1:8 (P47:actin). These activities were Ca independent. Purified TSP-labelled P47 lost the binding ability with membrane, also the inhibitory activity in actin polymerization. Therefore, they propose the hypothesis that unphosphorylated P47 may loosely bind with the inside of plasma membrane of platelet and inhibit actin polymerization as a modulator, when stimulated, protein Kinase C rapidly phosphorylate P47 and induce the activation of cytoskeletal network and subsequently release reaction.

  8. Phosphorylation of erythrocyte membrane liberates calcium

    SciTech Connect

    Chauhan, V.P.S.; Brockerhoff, H.

    1986-05-01

    Phosphorylation of permeabilized erythrocyte ghost membranes with ATP results in an increase free calcium level as measured with the help of Ca/sup 2 +/ electrode and /sup 45/Ca. This effect could not be observed in the presence of p/sup -/ chloromercuric benzoate, an inhibitor of kinases. The rise in the free calcium due to phosphorylation of the membrane was accompanied by a decrease in the level of phosphatidylinositol (PI) and an increase in phosphatidylinositolmonophosphate (PIP) and phosphatidylinositolbisphosphate (PIP/sub 2/). These results support the proposal that an inositol shuttle, PI in equilibrium PIP in equilibrium PIP/sub 2/, operates to maintain the intracellular calcium concentration. The cation is believed to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with the participation of both PO and fatty acid ester CO groups. When the inositol group of such a cage is phosphorylated, inter-headgroup hydrogen bonding between the lipids is broken. As a result the cage opens and calcium is released.

  9. Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

    PubMed Central

    O’Keefe, Rachel A.; Blice-Baum, Anna; Gong, Xuan; Karaca, Esra

    2016-01-01

    Abstract The fragile X mental retardation protein (FMRP) is an mRNA-binding regulator of protein translation that associates with 4-6% of brain transcripts and is central to neurodevelopment. Autism risk genes’ transcripts are overrepresented among FMRP-binding mRNAs, and FMRP loss-of-function mutations are responsible for fragile X syndrome, the most common cause of monogenetic autism. It is thought that FMRP-dependent translational repression is governed by the phosphorylation of serine residue 499 (S499). However, recent evidence suggests that S499 phosphorylation is not modulated by metabotropic glutamate receptor class I (mGluR-I) or protein phosphatase 2A (PP2A), two molecules shown to regulate FMRP translational repression. Moreover, the mammalian FMRP S499 kinase remains unknown. We found that casein kinase II (CK2) phosphorylates murine FMRP S499. Further, we show that phosphorylation of FMRP S499 permits phosphorylation of additional, nearby residues. Evidence suggests that these nearby residues are modulated by mGluR-I and PP2A pathways. These data support an alternative phosphodynamic model of FMRP that is harmonious with prior studies and serves as a framework for further investigation. PMID:27957526

  10. Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa

    SciTech Connect

    Kelly-Wintenberg, K.; Anderson, T.; Montie, T.C. )

    1990-09-01

    Purified flagella from two strains of {sup 32}P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined.

  11. Measurement of acylcarnitine substrate to product ratios specific to biotin-dependent carboxylases offers a combination of indicators of biotin status in humans.

    PubMed

    Bogusiewicz, Anna; Horvath, Thomas D; Stratton, Shawna L; Mock, Donald M; Boysen, Gunnar

    2012-09-01

    This work describes a novel liquid chromatography tandem MS (LC-MS/MS) method for the determination of ratios of acylcarnitines arising from acyl-CoA substrates and products that reflect metabolic disturbances caused by marginal biotin deficiency. The urinary ratios reflecting reduced activities of biotin-dependent enzymes include the following: 1) the ratio of 3-hydroxyisovalerylcarnitine : 3-methylglutarylcarnitine (3HIAc : MGc) for methylcrotonyl-CoA carboxylase; 2) the ratio of propionylcarnitine:methylmalonylcarnitine (Pc : MMc) for propionyl-CoA carboxylase (PCC); and 3) the ratio of acetylcarnitine : malonylcarnitine (Ac : Mc) for acetyl-CoA carboxylase. To demonstrate the suitability of the LC-MS/MS method for biomonitoring, we measured the 3 ratios for 7 healthy adults at various time points (d 0, 14, and 28) during the induction of marginal biotin through the consumption of egg white. The mean change in the Pc : MMc ratio relative to d 0 was 5.3-fold by d 14 (P = 0.0049) and 8.5-fold by d 28 (P = 0.0042). The mean change in the 3HIAc : MGc ratio was 2.8-fold by d 14 (P = 0.0022) and 3.8-fold by d 28 (P = 0.0001). The mean change in the Ac : Mc ratio was 2.9-fold by d 14 (P = 0.03) and 4.7-fold by d 28 (P = 0.02). The results suggest that simultaneous assessment of ratios of multiple biotin-dependent pathways offers insight into the complex metabolic disturbances caused by marginal biotin deficiency. We hypothesize that one or a combination of the ratios might be more sensitive or robust with respect to other nutrient deficiencies or confounding metabolic processes.

  12. A strategy to quantitate global phosphorylation of bone matrix proteins.

    PubMed

    Sroga, Grażyna E; Vashishth, Deepak

    2016-04-15

    Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured "in bulk" are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials.

  13. Complex kinase requirements for Chlamydia trachomatis Tarp phosphorylation.

    PubMed

    Mehlitz, Adrian; Banhart, Sebastian; Hess, Simone; Selbach, Matthias; Meyer, Thomas F

    2008-12-01

    Chlamydia trachomatis translocates the effector protein Tarp (translocated actin-recruiting phosphoprotein) into the host cell cytoplasm where it is quickly tyrosine phosphorylated. Abl and Src kinases have been implicated in Tarp phosphorylation; however, we observed that the situation is more complex. Chemical inhibition of Src family kinases confirmed a role for these kinases in Tarp phosphorylation. Infection of Src, Yes, Fyn (SYF)-deficient cells showed a dampened, but incompletely blocked, Tarp phosphorylation. Inhibition of Abl in an SYF background still did not completely block Tarp phosphorylation. Consequently, we tested additional kinases and found that Syk, but not Btk or Jak2, is a potent kinase of Tarp in vitro. Inhibition of Syk in an SYF background further blocked Tarp phosphorylation. Under these conditions, inclusion formation still proceeded normally. These data reveal a highly promiscuous substrate property of Tarp and set the stage for further functional characterization of Tarp phosphorylation during host cell infection.

  14. Multistep phosphorylation systems: tunable components of biological signaling circuits

    PubMed Central

    Valk, Evin; Venta, Rainis; Örd, Mihkel; Faustova, Ilona; Kõivomägi, Mardo; Loog, Mart

    2014-01-01

    Multisite phosphorylation of proteins is a powerful signal processing mechanism that plays crucial roles in cell division and differentiation as well as in disease. We recently demonstrated a novel phenomenon in cell cycle regulation by showing that cyclin-dependent kinase–dependent multisite phosphorylation of a crucial substrate is performed sequentially in the N-to-C terminal direction along the disordered protein. The process is controlled by key parameters, including the distance between phosphorylation sites, the distribution of serines and threonines in sites, and the position of docking motifs. According to our model, linear patterns of phosphorylation along disordered protein segments determine the signal-response function of a multisite phosphorylation switch. Here we discuss the general advantages and engineering principles of multisite phosphorylation networks as processors of kinase signals. We also address the idea of using the mechanistic logic of linear multisite phosphorylation networks to design circuits for synthetic biology applications. PMID:25368420

  15. Tyrosine phosphorylation of Rab7 by Src kinase.

    PubMed

    Lin, Xiaosi; Zhang, Jiaming; Chen, Lingqiu; Chen, Yongjun; Xu, Xiaohui; Hong, Wanjin; Wang, Tuanlao

    2017-03-20

    The small molecular weight GTPase Rab7 is a key regulator for late endosomal/lysosomal membrane trafficking, it was known that Rab7 is phosphorylated, but the corresponding kinase and the functional regulation of Rab7 phosphorylation remain unclear. We provide evidence here that Rab7 is a substrate of Src kinase, and is tyrosine-phosphorylated by Src, withY183 residue of Rab7 being the optimal phosphorylation site for Src. Further investigations demonstrated that the tyrosine phosphorylation of Rab7 depends on the guanine nucleotide binding activity of Rab7 and the activity of Src kinase. The tyrosine phosphorylation of Rab7 is physiologically induced by EGF, and impairs the interaction of Rab7 with RILP, consequently inhibiting EGFR degradation and sustaining Akt signaling. These results suggest that the tyrosine phosphorylation of Rab7 may be involved in coordinating membrane trafficking and cell signaling.

  16. Puzzling over protein cysteine phosphorylation--assessment of proteomic tools for S-phosphorylation profiling.

    PubMed

    Buchowiecka, A K

    2014-09-07

    Cysteine phosphorylation has recently been discovered in both prokaryotic and eukaryotic systems, and is thought to play crucial roles in signaling and regulation of cellular responses. This article explores the topics of chemical stability of this type of structural modification and the resulting issues regarding affinity enrichment of S-phosphopeptides and their mass spectrometry-based detection in the course of general proteomics studies. Together, this work suggests that the current advances in phosphoproteomic methodologies provide adequate tools for investigating protein cysteine phosphorylation and appear to be immediately available for practical implementation. The article provides useful information necessary for designing experiments in the emerging cysteine phosphoproteomics. The examples of methodological proposals for S-linked phosphorylation detection are included herein in order to stimulate development of new approaches by the phosphoproteomic community.

  17. Phosphorylated testis-specific serine/threonine kinase 4 may phosphorylate Crem at Ser-117.

    PubMed

    Fu, Guolong; Wei, Youheng; Wang, Xiaoli; Yu, Long

    2016-06-01

    We aimed to investigate the internal existence status of testis-specific serine/threonine kinase 4 (Tssk4) and the interaction of Tssk4 and Cre-responsive element modulator (Crem). The internal existence status of Tssk4 in testis of mice was detected using western blotting and dephosphorylation method. The interaction of Tssk4 and Crem was analyzed by western blotting, immunohistochemistry, immunofluorescence, in vitro co-immunoprecipitation assays, and in vitro kinase assay. The results revealed that Tssk4 existed in testis both in phosphorylation and unphosphorylation status by a temporal manner with the development of testis. Immunofluorescence results showed that Tssk4 had identical distribution pattern with Crem in testis, which was utterly different to the localization of Cre-responsive element binding (Creb). In conclusion, our study demonstrated that phosphorylated Tssk4 might participate in testis genes expressions by phosphorylating Crem at Ser-117.

  18. Transforming growth factor-{beta}-inducible phosphorylation of Smad3.

    PubMed

    Wang, Guannan; Matsuura, Isao; He, Dongming; Liu, Fang

    2009-04-10

    Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.

  19. Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.

    PubMed

    Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan M F; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul M K

    2015-08-01

    Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin

  20. Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

    PubMed Central

    Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan MF; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul MK

    2015-01-01

    Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of

  1. A kinetic study of the effects of phosphate and organic phosphates on the activity of phosphoenolpyruvate carboxylase from Crassula argentea.

    PubMed

    Meyer, C R; Rustin, P; Wedding, R T

    1989-05-15

    The effects of phosphate and several phosphate-containing compounds on the activity of purified phosphoenolpyruvate carboxylase (PEPC) from the crassulacean acid metabolism plant, Crassula argentea, were investigated. When assayed at subsaturating phosphoenolpyruvate (PEP) concentrations, low concentrations of most of the compounds tested were found to stimulate PEPC activity. This activation, variable in extent, was found in all cases to be competitive with glucose 6-phosphate (Glc-6-P) stimulation, suggesting that these effectors bind to the Glc-6-P site. At higher concentrations, depending upon the effector molecule studied, deactivation, inhibition, or no response was observed. More detailed studies were performed with Glc-6-P, AMP, phosphoglycolate, and phosphate. AMP had previously been shown to be a specific ligand for the Glc-6-P site. The main effect of Glc-6-P and AMP on the kinetic parameters was to decrease the apparent Km and increase Vmax/Km. AMP also caused a decrease in the Vmax of the reaction. In contrast, phosphoglycolate acted essentially as a competitive inhibitor increasing the apparent Km for PEP and decreasing Vmax/Km. Inorganic phosphate had a biphasic effect on the kinetic parameters, resulting in a transient decrease in Km followed by an increase of the apparent Km for PEP with increasing concentration of phosphate. The Vmax also was decreased with increasing phosphate concentrations. Further, the enzyme appeared to respond to the complex of phosphate with magnesium. In the presence of a saturating concentration of AMP, no activation but rather inhibition was observed with increasing phosphate concentration. This is consistent with the binding of phosphate to two separate sites--the Glc-6-P activation site and an inhibitory site, a phenomenon that may be occurring with other phosphate containing compounds. High concentrations of phosphate with magnesium were found to protect enzyme activity when PEPC, previously shown to contain an

  2. Genetic Manipulation of Neurofilament Protein Phosphorylation.

    PubMed

    Jones, Maria R; Villalón, Eric; Garcia, Michael L

    2016-01-01

    Neurofilament biology is important to understanding structural properties of axons, such as establishment of axonal diameter by radial growth. In order to study the function of neurofilaments, a series of genetically modified mice have been generated. Here, we describe a brief history of genetic modifications used to study neurofilaments, as well as an overview of the steps required to generate a gene-targeted mouse. In addition, we describe steps utilized to analyze neurofilament phosphorylation status using immunoblotting. Taken together, these provide comprehensive analysis of neurofilament function in vivo, which can be applied to many systems.

  3. Moringa oleifera leaf extract ameliorates alloxan-induced diabetes in rats by regeneration of β cells and reduction of pyruvate carboxylase expression.

    PubMed

    Abd El Latif, Amira; El Bialy, Badr El Said; Mahboub, Hamada Dahi; Abd Eldaim, Mabrouk Attia

    2014-10-01

    Moringa oleifera Lam. contains many active ingredients with nutritional and medicinal values. It is commonly used in folk medicine as an antidiabetic agent. The present study was designed to investigate how an aqueous extract from the leaves of M. oleifera reveals hypoglycemia in diabetic rats. M. oleifera leaf extract counteracted the alloxan-induced diabetic effects in rats as it normalized the elevated serum levels of glucose, triglycerides, cholesterol, and malondialdehyde, and normalized mRNA expression of the gluconeogenic enzyme pyruvate carboxylase in hepatic tissues. It also increased live body weight gain and normalized the reduced mRNA expression of fatty acid synthase in the liver of diabetic rats. Moreover, it restored the normal histological structure of the liver and pancreas damaged by alloxan in diabetic rats. This study revealed that the aqueous extract of M. oleifera leaves possesses potent hypoglycemic effects through the normalization of elevated hepatic pyruvate carboxylase enzyme and regeneration of damaged hepatocytes and pancreatic β cells via its antioxidant properties.

  4. Mild water stress effects on carbon-reduction-cycle intermediates, ribulose bisphosphate carboxylase activity, and spatial homogeneity of photosynthesis in intact leaves

    SciTech Connect

    Sharkey, T.D.; Seemann, J.R. Univ. of Nevada, Reno )

    1989-04-01

    We have examined the effect of mild water stress on photosynthetic chloroplast reactions of intact Phaseolus vulgaris leaves by measuring two parameters of ribulose bisphosphate (RuBP) carboxylase activity and the pool sizes of RuBP, 3-phosphoglycerate (PGA), triose phosphates, hexose monophosphates, and ATP. We also tested for patchy stomatal closure by feeding {sup 14}CO{sub 2}. The k{sub cat} of RuBP carboxylase (moles CO{sub 2} fixed per mole enzyme per second) which could be measured after incubating the enzyme with CO{sub 2} and Mg{sup 2+} was unchanged by water stress. The ratio of activity before and after incubation with CO{sub 2} and Mg{sup 2+} (the carbamylation state) was slightly reduced by severe stress but not by mild stress. Likewise, the concentration of RuBP was slightly reduced by severe stress but not by mild stress. The concentration of PGA was markedly reduced by both mild and severe water stress. The concentration of triose phosphates did not decline as much as PGA. We found that photosynthesis in water stressed leaves occurred in patches. The patchiness of photosynthesis during water stress may lead to an underestimation of the effect of stomatal closure. We conclude that reductions in whole leaf photosynthesis caused by mild water stress are primarily the result of stomatal closure and that there is no indication of damage to chloroplast reactions.

  5. A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units

    PubMed Central

    Ray, Lauren; Valentic, Timothy R.; Miyazawa, Takeshi; Withall, David M.; Song, Lijiang; Milligan, Jacob C.; Osada, Hiroyuki; Takahashi, Shunji; Tsai, Shiou-Chuan; Challis, Gregory L.

    2016-01-01

    Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues. PMID:28000660

  6. A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units

    NASA Astrophysics Data System (ADS)

    Ray, Lauren; Valentic, Timothy R.; Miyazawa, Takeshi; Withall, David M.; Song, Lijiang; Milligan, Jacob C.; Osada, Hiroyuki; Takahashi, Shunji; Tsai, Shiou-Chuan; Challis, Gregory L.

    2016-12-01

    Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.

  7. Regulation of cardiac C-protein phosphorylation

    SciTech Connect

    Titus, F.L.

    1985-01-01

    Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased (/sup 32/P)phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and (/sup 32/P)phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 ..mu..M Iso and 17% in hearts exposed to Iso plus 1 ..mu..M methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed.

  8. Modelling the Krebs cycle and oxidative phosphorylation.

    PubMed

    Korla, Kalyani; Mitra, Chanchal K

    2014-01-01

    The Krebs cycle and oxidative phosphorylation are the two most important sets of reactions in a eukaryotic cell that meet the major part of the total energy demands of a cell. In this paper, we present a computer simulation of the coupled reactions using open source tools for simulation. We also show that it is possible to model the Krebs cycle with a simple black box with a few inputs and outputs. However, the kinetics of the internal processes has been modelled using numerical tools. We also show that the Krebs cycle and oxidative phosphorylation together can be combined in a similar fashion - a black box with a few inputs and outputs. The Octave script is flexible and customisable for any chosen set-up for this model. In several cases, we had no explicit idea of the underlying reaction mechanism and the rate determining steps involved, and we have used the stoichiometric equations that can be easily changed as and when more detailed information is obtained. The script includes the feedback regulation of the various enzymes of the Krebs cycle. For the electron transport chain, the pH gradient across the membrane is an essential regulator of the kinetics and this has been modelled empirically but fully consistent with experimental results. The initial conditions can be very easily changed and the simulation is potentially very useful in a number of cases of clinical importance.

  9. Control of Collagen Triple Helix Stability by Phosphorylation.

    PubMed

    Acevedo-Jake, Amanda M; Ngo, Daniel H; Hartgerink, Jeffrey D

    2017-03-10

    The phosphorylation of the collagen triple helix plays an important role in collagen synthesis, assembly, signaling, and immune response, although no reports detailing the effect this modification has on the structure and stability of the triple helix exist. Here we investigate the changes in stability and structure resulting from the phosphorylation of collagen. Additionally, the formation of pairwise interactions between phosphorylated residues and lysine is examined. In all tested cases, phosphorylation increases helix stability. When charged-pair interactions are possible, stabilization via phosphorylation can play a very large role, resulting inasmuch as a 13.0 °C increase in triple helix stability. Two-dimensional NMR and molecular modeling are used to study the local structure of the triple helix. Our results suggest a mechanism of action for phosphorylation in the regulation of collagen and also expand upon our understanding of pairwise amino acid stabilization of the collagen triple helix.

  10. Phosphorylation modifies the molecular stability of β-amyloid deposits

    PubMed Central

    Rezaei-Ghaleh, Nasrollah; Amininasab, Mehriar; Kumar, Sathish; Walter, Jochen; Zweckstetter, Markus

    2016-01-01

    Protein aggregation plays a crucial role in neurodegenerative diseases. A key feature of protein aggregates is their ubiquitous modification by phosphorylation. Little is known, however, about the molecular consequences of phosphorylation of protein aggregates. Here we show that phosphorylation of β-amyloid at serine 8 increases the stability of its pathogenic aggregates against high-pressure and SDS-induced dissociation. We further demonstrate that phosphorylation results in an elevated number of hydrogen bonds at the N terminus of β-amyloid, the region that is critically regulated by a variety of post-translational modifications. Because of the increased lifetime of phosphorylated β-amyloid aggregates, phosphorylation can promote the spreading of β-amyloid in Alzheimer pathogenesis. Our study suggests that regulation of the molecular stability of protein aggregates by post-translational modifications is a crucial factor for disease progression in the brain. PMID:27072999

  11. Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases.

    PubMed

    Jewett, Travis J; Dooley, Cheryl A; Mead, David J; Hackstadt, Ted

    2008-06-27

    The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.

  12. Phosphorylation modifies the molecular stability of β-amyloid deposits

    NASA Astrophysics Data System (ADS)

    Rezaei-Ghaleh, Nasrollah; Amininasab, Mehriar; Kumar, Sathish; Walter, Jochen; Zweckstetter, Markus

    2016-04-01

    Protein aggregation plays a crucial role in neurodegenerative diseases. A key feature of protein aggregates is their ubiquitous modification by phosphorylation. Little is known, however, about the molecular consequences of phosphorylation of protein aggregates. Here we show that phosphorylation of β-amyloid at serine 8 increases the stability of its pathogenic aggregates against high-pressure and SDS-induced dissociation. We further demonstrate that phosphorylation results in an elevated number of hydrogen bonds at the N terminus of β-amyloid, the region that is critically regulated by a variety of post-translational modifications. Because of the increased lifetime of phosphorylated β-amyloid aggregates, phosphorylation can promote the spreading of β-amyloid in Alzheimer pathogenesis. Our study suggests that regulation of the molecular stability of protein aggregates by post-translational modifications is a crucial factor for disease progression in the brain.

  13. Chemical Approaches to Studying Labile Amino Acid Phosphorylation.

    PubMed

    Marmelstein, Alan M; Moreno, Javier; Fiedler, Dorothea

    2017-04-01

    Phosphorylation of serine, threonine, and tyrosine residues is the archetypal posttranslational modification of proteins. While phosphorylation of these residues has become standard textbook knowledge, phosphorylation of other amino acid side chains is underappreciated and minimally characterized by comparison. This disparity is rooted in the relative instability of these chemically distinct amino acid side chain moieties, namely phosphoramidates, acyl phosphates, thiophosphates, and phosphoanhydrides. In the case of the O-phosphorylated amino acids, synthetic constructs were critical to assessing their stability and developing tools for their study. As the chemical biology community has become more aware of these alternative phosphorylation sites, methodology has been developed for the synthesis of well-characterized standards and close mimics of these phosphorylated amino acids as well. In this article, we review the synthetic chemistry that is a prerequisite to progress in this field.

  14. Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides

    NASA Astrophysics Data System (ADS)

    Bertran-Vicente, Jordi; Penkert, Martin; Nieto-Garcia, Olaia; Jeckelmann, Jean-Marc; Schmieder, Peter; Krause, Eberhard; Hackenberger, Christian P. R.

    2016-09-01

    In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICBGlc, which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes.

  15. Tyrosine phosphorylation of clathrin heavy chain under oxidative stress.

    PubMed

    Ihara, Yoshito; Yasuoka, Chie; Kageyama, Kan; Wada, Yoshinao; Kondo, Takahito

    2002-09-20

    In mouse pancreatic insulin-producing betaTC cells, oxidative stress due to H(2)O(2) causes tyrosine phosphorylation in various proteins. To identify proteins bearing phosphotyrosine under stress, the proteins were affinity purified using an anti-phosphotyrosine antibody-conjugated agarose column. A protein of 180kDa was identified as clathrin heavy chain (CHC) by electrophoresis and mass spectrometry. Immunoprecipitated CHC showed tyrosine phosphorylation upon H(2)O(2) treatment and the phosphorylation was suppressed by the Src kinase inhibitor, PP2. The phosphorylation status of CHC affected the intracellular localization of CHC and the clathrin-dependent endocytosis of transferrin under oxidative stress. In conclusion, CHC is a protein that is phosphorylated at tyrosine by H(2)O(2) and this phosphorylation status is implicated in the intracellular localization and functions of CHC under oxidative stress. The present study demonstrates that oxidative stress affects intracellular vesicular trafficking via the alteration of clathrin-dependent vesicular trafficking.

  16. Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo.

    PubMed

    Chang, Audrey N; Battiprolu, Pavan K; Cowley, Patrick M; Chen, Guohua; Gerard, Robert D; Pinto, Jose R; Hill, Joseph A; Baker, Anthony J; Kamm, Kristine E; Stull, James T

    2015-04-24

    In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca(2+) sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity.

  17. Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria.

    PubMed

    Gnad, Florian; Forner, Francesca; Zielinska, Dorota F; Birney, Ewan; Gunawardena, Jeremy; Mann, Matthias

    2010-12-01

    High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the

  18. The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser11 in developing castor oil seeds.

    PubMed

    Fedosejevs, Eric T; Gerdis, Suzanne A; Ying, Sheng; Pyc, Michal; Anderson, Erin M; Snedden, Wayne A; Mullen, Robert T; She, Yi-Min; Plaxton, William C

    2016-10-15

    Imported sucrose is cleaved by sucrose synthase (SUS) as a critical initial reaction in the biosynthesis of storage end-products by developing seeds. Although SUS is phosphorylated at a conserved seryl residue by an apparent CDPK (Ca(2+)-dependent protein kinase) in diverse plant tissues, the functions and mechanistic details of this process remain obscure. Thus, the native CDPK that phosphorylates RcSUS1 (Ricinus communis SUS1) at Ser(11) in developing COS (castor oil seeds) was highly purified and identified as RcCDPK2 by MS/MS. Purified RcSUS1-K (-kinase) and heterologously expressed RcCDPK2 catalyzed Ca(2+)-dependent Ser(11) phosphorylation of RcSUS1 and its corresponding dephosphopeptide, while exhibiting a high affinity for free Ca(2+) ions [K0.5(Ca(2+)) < 0.4 µM]. RcSUS1-K activity, RcCDPK2 expression, and RcSUS1 Ser(11) phosphorylation peaked during early COS development and then declined in parallel. The elimination of sucrose import via fruit excision triggered RcSUS1 dephosphorylation but did not alter RcSUS1-K activity, suggesting a link between sucrose signaling and posttranslational RcCDPK2 control. Both RcCDPK2-mCherry and RcSUS1-EYFP co-localized throughout the cytosol when transiently co-expressed in tobacco suspension cells, although RcCDPK2-mCherry was also partially localized to the nucleus. Subcellular fractionation revealed that ∼20% of RcSUS1-K activity associates with microsomal membranes in developing COS, as does RcSUS1. In contrast with RcCDPK1, which catalyzes inhibitory phosphorylation of COS bacterial-type phosphoenolpyruvate carboxylase at Ser(451), RcCDPK2 exhibited broad substrate specificity, a wide pH-activity profile centered at pH 8.5, and insensitivity to metabolite effectors or thiol redox status. Our combined results indicate a possible link between cytosolic Ca(2+)-signaling and the control of photosynthate partitioning during COS development.

  19. Sequential Phosphorylation of Smoothened Transduces Graded Hedgehog Signaling

    PubMed Central

    Su, Ying; Ospina, Jason K.; Zhang, Junzheng; Michelson, Andrew P.; Schoen, Adam M.; Zhu, Alan Jian

    2012-01-01

    The correct interpretation of a gradient of the morphogen Hedgehog (Hh) during development requires phosphorylation of the Hh signaling activator Smoothened (Smo); however, the molecular mechanism by which Smo transduces graded Hh signaling is not well understood. We show that regulation of the phosphorylation status of Smo by distinct phosphatases at specific phosphorylated residues creates differential thresholds of Hh signaling. Phosphorylation of Smo was initiated by adenosine 3′,5′-monophosphate (cAMP)–dependent protein kinase (PKA) and further enhanced by casein kinase I (CKI). We found that protein phosphatase 1 (PP1) directly dephosphorylated PKA-phosphorylated Smo to reduce signaling mediated by intermediate concentrations of Hh, whereas PP2A specifically dephosphorylated PKA-primed, CKI-phosphorylated Smo to restrict signaling by high concentrations of Hh. We also established a functional link between sequentially phosphorylated Smo species and graded Hh activity. Thus, we propose a sequential phosphorylation model in which precise interpretation of morphogen concentration can be achieved upon versatile phosphatase-mediated regulation of the phosphorylation status of an essential activator in developmental signaling. PMID:21730325

  20. Mapping of phosphorylation sites in polyomavirus large T antigen

    SciTech Connect

    Hassauer, M.; Scheidtmann, K.H.; Walter, G.

    1986-06-01

    The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, /sup 32/P/sub i/-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed.

  1. Two Pdk1 phosphorylation sites on the plant cell death suppressor Adi3 contribute to substrate phosphorylation

    PubMed Central

    Gray, Joel W.; Nelson Dittrich, Anna C.; Chen, Sixue; Avila, Julian; Giavalisco, Patrick; Devarenne, Timothy P.

    2015-01-01

    The tomato AGC kinase Adi3 is phosphorylated by Pdk1 for activation of its cell death suppression activity. The Pdk1 phosphorylation site for activation of Adi3 is at Ser539. However, there is at least one additional Pdk1 phosphorylation site on Adi3 that has an unknown function. Here we identify an Arabidopsis thaliana sequence homologue of Adi3 termed AGC1-3. Two Pdk1 phosphorylation sites were identified on AGC1-3, activation site Ser596 and Ser269, and by homology Ser212 on Adi3 was identified as a second Pdk1 phosphorylation site. While Ser212 is not required for Adi3 autophosphorylation, Ser212 was shown to be required for full phosphorylation of the Adi3 substrate Gal83. PMID:23507047

  2. A coulombic hypothesis of mitochondrial oxidative phosphorylation.

    PubMed

    Malpress, F H

    1984-08-21

    A coulombic hypothesis of mitochondrial oxidative phosphorylation is presented, founded upon the evidence for negative fixed charge formation during electron transport chain activity. The intermediary force is electrostatic (psi H) and not electrochemical (delta mu H). The electrochemical potential of the chemiosmotic hypothesis is identified as a "phantom" parameter which owes its delusive existence to the procedures by which it is measured. The connection between psi H and the conditional delta mu H values is examined; it entails the use of a variable conversion factor, f, where delta mu H (mV) = f psi H, and the concept of the "protonic status" of the diffuse double layer. A number of problems which beset the chemiosmotic view are reappraised in the light of the new interpretation, and find authentic solutions.

  3. The regulation of STIM1 by phosphorylation

    PubMed Central

    Pozo-Guisado, Eulalia; Martin-Romero, Francisco Javier

    2013-01-01

    Calcium ion (Ca2+) concentration plays a key role in cell signaling in eukaryotic cells. At the cellular level, Ca2+ directly participates in such diverse cellular events as adhesion and migration, differentiation, contraction, secretion, synaptic transmission, fertilization, and cell death. As a consequence of these diverse actions, the cytosolic concentration of free Ca2+ is tightly regulated by the coordinated activity of Ca2+ channels, Ca2+ pumps, and Ca2+-binding proteins. Although many of these regulators have been studied in depth, other proteins have been described recently, and naturally far less is known about their contribution to cell physiology. Within this last group of proteins, STIM1 has emerged as a major contributor to Ca2+ signaling by means of its activity as Ca2+ channel regulator. STIM1 is a protein resident mainly, but not exclusively, in the endoplasmic reticulum (ER), and activates a set of plasma membrane Ca2+ channels termed store-operated calcium channels (SOCs) when the concentration of free Ca2+ within the ER drops transiently as a result of Ca2+ release from this compartment. Knowledge regarding the molecular architecture of STIM1 has grown considerably during the last years, and several structural domains within STIM1 have been reported to be required for the specific molecular interactions with other important players in Ca2+ signaling, such as Ca2+ channels and microtubules. Within the modulators of STIM1, phosphorylation has been shown to both activate and inactivate STIM1-dependent Ca2+ entry depending on the cell type, cell cycle phase, and the specific residue that becomes modified. Here we shall review current knowledge regarding the modulation of STIM1 by phosphorylation. PMID:24505502

  4. Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts.

    PubMed

    Zhang, H; Hoff, H; Marinucci, T; Cristofalo, V J; Sell, C

    2000-08-25

    The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.

  5. PKCβ–dependent phosphorylation of the glycine transporter 1

    PubMed Central

    Vargas-Medrano, Javier; Castrejon-Tellez, Vicente; Fernando, Plenge; Ramirez, Ivan; Miranda, Manuel

    2011-01-01

    The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [32P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23-40% -inhibition on Vmax was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that phosphorylation that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation. PMID:21864610

  6. A grammar inference approach for predicting kinase specific phosphorylation sites.

    PubMed

    Datta, Sutapa; Mukhopadhyay, Subhasis

    2015-01-01

    Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner.

  7. Cloning of a chicken liver cDNA encoding 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase by functional complementation of Escherichia coli pur mutants.

    PubMed Central

    Chen, Z D; Dixon, J E; Zalkin, H

    1990-01-01

    We have used functional complementation of Escherichia coli pur mutants to clone avian cDNA encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase-5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase, the bifunctional enzyme catalyzing steps 6 and 7 in the pathway for de novo purine nucleotide synthesis. Mutational analyses have been used to establish the structure-function relationship: NH2-SAICAR synthetase-AIR carboxylase-COOH. The amino acid sequence of the SAICAR synthetase domain is homologous to that of bacterial purC-encoded enzymes, and the sequence of the following AIR carboxylase domain is homologous to that of bacterial purE-encoded enzymes. In E. coli, AIR carboxylase is the product of genes purEK with the purK subunit postulated to have a role in CO2 binding. The avian enzyme lacks sequences corresponding to purK yet functions in E. coli. Functional complementation of E. coli pur mutants can be used to clone additional avian cDNAs for de novo purine nucleotide synthesis. Images PMID:1691501

  8. Simple determination of the CO sub 2 /O sub 2 specificity of Ribulose-1,5-bisphosphate carboxylase/oxygenase by the specific radioactivity of ( sup 14 C) glycerate 3-phosphate

    SciTech Connect

    Genhai Zhu; Jensen, R.G.; Hallick, R.B.; Wildner, G.F. )

    1992-02-01

    A new method is presented for measurement of the CO{sub 2}/O{sub 2} specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The ({sup 14}C)3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. {sup 14}CO{sub 2} fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO{sub 2} in O{sub 2}-saturated water and carboxylase only with 160 micromolar CO{sub 2} under N{sub 2}. Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the ({sup 14}C)PGA in the same peak. The specificity factor of Rubisco from spinach (Spinacia oleracea L.) (93 {plus minus} 4), from the green alga Chlamydomonas reinhardtii (66 {plus minus} 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods.

  9. The role of the VQIVYK peptide in tau protein phosphorylation.

    PubMed

    Perez, Mar; Santa-María, Ismael; Tortosa, Elena; Cuadros, Raquel; Del Valle, Mercedes; Hernández, Felix; Moreno, Francisco J; Avila, Jesús

    2007-11-01

    Although it remains unclear whether they are related to one another, tau aggregation and phosphorylation are the main pathological hallmarks of the neuronal disorders known as tauopathies. The capacity to aggregate is impaired in a variant of the tau 3R isoform that lacks residues 306-311 (nomenclature for the largest CNS tau isoform) and hence, we have taken advantage of this feature to study how phosphorylation and aggregation may be related as well as the role of this six amino acid peptide (VQIVYK). Through these analyses, we found that the phosphorylation of the tau variant was higher than that of the complete tau protein and that not only the deletion of these residues, but also the interaction of these residues, in tau 3R, with thioflavin-S augmented tau phosphorylation by glycogen synthase kinase 3. In addition, the binding of the peptide containing the residues 306-311 to the whole tau protein provoked an increase in tau phosphorylation. This observation could be physiologically relevant as may suggest that tau-tau interactions, through those residues, facilitate tau phosphorylation. In summary, our data indicate that deletion of residues VQIVYK, in tau protein produces an increase in tau phosphorylation, without tau aggregation, because the VQIVYK peptide, that favors aggregation, is missing. On the other hand, when the whole tau protein interacts with thioflavin-S or the peptide VQIVYK, an increase in both aggregation and phosphorylation occurs.

  10. Anillin Phosphorylation Controls Timely Membrane Association and Successful Cytokinesis

    PubMed Central

    Kim, Hyunjung; Johnson, James M.; Brahma, Sarang; Burkard, Mark E.

    2017-01-01

    During cytokinesis, a contractile ring generates the constricting force to divide a cell into two daughters. This ring is composed of filamentous actin and the motor protein myosin, along with additional structural and regulatory proteins, including anillin. Anillin is a required scaffold protein that links the actomyosin ring to membrane and its organizer, RhoA. However, the molecular basis for timely action of anillin at cytokinesis remains obscure. Here, we find that phosphorylation regulates efficient recruitment of human anillin to the equatorial membrane. Anillin is highly phosphorylated in mitosis, and is a substrate for mitotic kinases. We surveyed function of 46 residues on anillin previously found to be phosphorylated in human cells to identify those required for cytokinesis. Among these sites, we identified S635 as a key site mediating cytokinesis. Preventing S635 phosphorylation adjacent to the AH domain disrupts anillin concentration at the equatorial cortex at anaphase, whereas a phosphomimetic mutant, S635D, partially restores this localization. Time-lapse videomicroscopy reveals impaired recruitment of S635A anillin to equatorial membrane and a transient unstable furrow followed by ultimate failure in cytokinesis. A phosphospecific antibody confirms phosphorylation at S635 in late cytokinesis, although it does not detect phosphorylation in early cytokinesis, possibly due to adjacent Y634 phosphorylation. Together, these findings reveal that anillin recruitment to the equatorial cortex at anaphase onset is enhanced by phosphorylation and promotes successful cytokinesis. PMID:28081137

  11. Importance of tyrosine phosphorylation in receptor kinase complexes.

    PubMed

    Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

    2015-05-01

    Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants.

  12. Altered protein phosphorylation as a resource for potential AD biomarkers

    PubMed Central

    Henriques, Ana Gabriela; Müller, Thorsten; Oliveira, Joana Machado; Cova, Marta; da Cruz e Silva, Cristóvão B.; da Cruz e Silva, Odete A. B.

    2016-01-01

    The amyloidogenic peptide, Aβ, provokes a series of events affecting distinct cellular pathways regulated by protein phosphorylation. Aβ inhibits protein phosphatases in a dose-dependent manner, thus it is expected that the phosphorylation state of specific proteins would be altered in response to Aβ. In fact several Alzheimer’s disease related proteins, such as APP and TAU, exhibit pathology associated hyperphosphorylated states. A systems biology approach was adopted and the phosphoproteome, of primary cortical neuronal cells exposed to Aβ, was evaluated. Phosphorylated proteins were recovered and those whose recovery increased or decreased, upon Aβ exposure across experimental sets, were identified. Significant differences were evident for 141 proteins and investigation of their interactors revealed key protein clusters responsive to Aβ treatment. Of these, 73 phosphorylated proteins increased and 68 decreased upon Aβ addition. These phosphorylated proteins represent an important resource of potential AD phospho biomarkers that should be further pursued. PMID:27466139

  13. Altered protein phosphorylation as a resource for potential AD biomarkers.

    PubMed

    Henriques, Ana Gabriela; Müller, Thorsten; Oliveira, Joana Machado; Cova, Marta; da Cruz E Silva, Cristóvão B; da Cruz E Silva, Odete A B

    2016-07-28

    The amyloidogenic peptide, Aβ, provokes a series of events affecting distinct cellular pathways regulated by protein phosphorylation. Aβ inhibits protein phosphatases in a dose-dependent manner, thus it is expected that the phosphorylation state of specific proteins would be altered in response to Aβ. In fact several Alzheimer's disease related proteins, such as APP and TAU, exhibit pathology associated hyperphosphorylated states. A systems biology approach was adopted and the phosphoproteome, of primary cortical neuronal cells exposed to Aβ, was evaluated. Phosphorylated proteins were recovered and those whose recovery increased or decreased, upon Aβ exposure across experimental sets, were identified. Significant differences were evident for 141 proteins and investigation of their interactors revealed key protein clusters responsive to Aβ treatment. Of these, 73 phosphorylated proteins increased and 68 decreased upon Aβ addition. These phosphorylated proteins represent an important resource of potential AD phospho biomarkers that should be further pursued.

  14. Functional determinants in transit sequences: import and partial maturation by vascular plant chloroplasts of the ribulose-1,5- bisphosphate carboxylase small subunit of Chlamydomonas

    PubMed Central

    1985-01-01

    The precursor of the ribulose-1,5-bisphosphate carboxylase small subunit and other proteins from Chlamydomonas reinhardtii are efficiently transported into chloroplasts isolated from spinach and pea. Thus, similar determinants specify precursor-chloroplast interactions in the alga and vascular plants. Removal of all or part of its transit sequence was found to block import of the algal small subunit into isolated chloroplasts. Comparison of available sequences revealed a nine amino acid segment conserved in the transit sequences of all small subunit precursors. A protease in the vascular plant chloroplasts recognized this region in the Chlamydomonas precursor and produced an intermediate form of the small subunit. We propose that processing of the small subunit precursor involves at least two proteolytic events; only one of these has been evolutionarily conserved. PMID:3965471

  15. Accumulation fatty acids of in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense

    NASA Astrophysics Data System (ADS)

    Leyva, Luis A.; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E.

    2014-10-01

    The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae.

  16. Five palmitoylated polypeptides in the 50 KDa range are not recognized by an antibody against ribulose-biphosphate-carboxylase-oxygenase in Chlamydomonas reinhardtii.

    PubMed

    Picaud, A; Hours, M C; Trémolières, A

    1993-11-30

    After incubation of Chlamydomonas reinhardtii cells with radioactive palmitic acid several labelled bands appeared after gel electrophoresis of delipidated protein extract. Among them, two bands (a major and a minor one) were detected in the 50 KDa range, which is the region where the LSU of the Rubisco (large sub-unit of the ribulose-biphosphate-carboxylase-oxygenase) was also found. Careful analyses by two-dimensional gel electrophoresis have shown that the five palmitate-labelled polypeptides detected in this region do not match with polypeptides immunoreacting with antibody against Rubisco. In addition, polypeptides labelled by palmitate cannot be immunoprecipitated with the same antibody further demonstrating that, in C. reinhardtii, the large sub-unit of Rubisco is not palmitoylated but unindentified proteins.

  17. Acetyl-coenzyme A carboxylase α gene variations may be associated with the direct effects of some antipsychotics on triglyceride levels

    PubMed Central

    Diaz, Francisco J.; Meary, Alexander; Arranz, Maria J.; Ruaño, Gualberto; Windemuth, Andreas; de Leon, Jose

    2009-01-01

    Acetyl-coenzyme A carboxylase α (ACACA) single-nucleotide polymorphism (SNP) (rs2229416) was significantly associated with hypertriglyceridemia, during exploration of antipsychotic direct effects on lipids. Neuropeptide Y (NPY) gene (rs1468271) and ACACB gene (rs2241220) SNPs were significantly associated with severe hypercholesterolemia. In the same sample (173 patients on olanzapine, quetiapine, chlorpromazine or mirtazapine [increasing the risk of hyperlipidemia] and 184 controls taking other antipsychotics), three (rs1266175, rs12453407 and rs9906543) of eight additional ACACA SNPs were significantly associated with hypertriglyceridemia in those taking drugs of interest, but not in controls. Five other ACACA SNPs, three additional NPY SNPs, or seven additional ACACB SNPs were not significant. PMID:19846279

  18. Direct and selective small-molecule inhibition of photosynthetic PEP carboxylase: New approach to combat C4 weeds in arable crops.

    PubMed

    Paulus, Judith Katharina; Förster, Kerstin; Groth, Georg

    2014-06-05

    Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C4 photosynthesis. Besides, non-photosynthetic isoforms of PEPC are found in bacteria and all types of plants, although not in animals or fungi. A single residue in the allosteric feedback inhibitor site of PEPC was shown to adjust the affinity of the photosynthetic and non-photosynthetic isoforms for feedback inhibition by metabolites of the C4 pathway. Here, we applied computational screening and biochemical analyses to identify molecules that selectively inhibit C4 PEPC, but have no effect on the activity of non-photosynthetic PEPCs. We found two types of selective inhibitors, catechins and quinoxalines. Binding constants in the lower μM range and a strong preference for C4 PEPC qualify the quinoxaline compounds as potential selective herbicides to combat C4 weeds.

  19. Determination of 3-hydroxyisovalerylcarnitine and other acylcarnitine levels using liquid chromatography-tandem mass spectrometry in serum and urine of a patient with multiple carboxylase deficiency.

    PubMed

    Maeda, Yasuhiro; Ito, Tetsuya; Ohmi, Hironori; Yokoi, Kyoko; Nakajima, Yoko; Ueta, Akihito; Kurono, Yukihisa; Togari, Hajime; Sugiyama, Naruji

    2008-07-15

    Due to its increased concentration in blood, 3-hydroxyisovalerylcarnitine (C5OH-I) is an important indicator for the diagnosis of organic acidemias in newborns. However, C5OH-I has not been used as a standard in tandem mass spectrometric (MS/MS) assays because its isolation is difficult. We developed a new synthesis of C5OH-I and investigated its behavior by MS/MS. A method using the multiple reaction monitoring (MRM) mode of MS/MS with HPLC was developed which provides high accuracy, precision and reproducibility. Acylcarnitine profiles in the serum and urine of a patient with multiple carboxylase deficiency (MCD) showed increased levels compared to a healthy patient.

  20. Oxidative Stress Induces Partial Degradation of the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Isolated Chloroplasts of Barley.

    PubMed Central

    Desimone, M.; Henke, A.; Wagner, E.

    1996-01-01

    The effects of oxidative stress on the degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were studied in isolated chloroplasts from barley (Hordeum vulgare L. cv Angora). Active oxygen (AO) was generated by varying the light intensity, the oxygen concentration, or the addition of herbicides or ADP-FeCl3-ascorbate to the medium. Oxidative treatments stimulated association of Rubisco with the insoluble fraction of chloroplasts and partial proteolysis of the large subunit (LSU). The most prominent degradation product of the LSU of Rubisco showed an apparent molecular mass of 36 kD. The data suggest that an increase in the amount of AO photogenerated by O2 reduction at photosystem I triggers Rubisco degradation. A possible relationship between AO-mediated denaturation of Rubisco and proteolysis of the LSU is discussed. PMID:12226330

  1. Genes encoding the alpha-carboxyltransferase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution.

    PubMed

    Li, Zhi-Guo; Yin, Wei-Bo; Guo, Huan; Song, Li-Ying; Chen, Yu-Hong; Guan, Rong-Zhan; Wang, Jing-Qiao; Wang, Richard R-C; Hu, Zan-Min

    2010-05-01

    Heteromeric acetyl coenzyme A carboxylase (ACCase), a rate-limiting enzyme in fatty acid biosynthesis in dicots, is a multi-enzyme complex consisting of biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase (alpha-CT and beta-CT). In the present study, four genes encoding alpha-CT were cloned from Brassica napus, and two were cloned from each of the two parental species, B. rapa and B. oleracea. Comparative and cluster analyses indicated that these genes were divided into two major groups. The major divergence between group-1 and group-2 occurred in the second intron. Group-2 alpha-CT genes represented the ancestral form in the genus Brassica. The divergence of group-1 and group-2 genes occurred in their common ancestor 12.96-17.78 million years ago (MYA), soon after the divergence of Arabidopsis thaliana and Brassica (15-20 MYA). This time of divergence is identical to that reported for the paralogous subgenomes of diploid Brassica species (13-17 MYA). Real-time reverse transcription PCR revealed that the expression patterns of the two groups of genes were similar in different organs, except in leaves. To better understand the regulation and evolution of alpha-CT genes, promoter regions from two sets of orthologous gene copies from B. napus, B. rapa, and B. oleracea were cloned and compared. The function of the promoter of gene Bnalpha-CT-1-1 in group-1 and gene Bnalpha-CT-2-1 in group-2 was examined by assaying beta-glucuronidase activity in transgenic A. thaliana. Our results will be helpful in elucidating the evolution and regulation of ACCase in oilseed rape.

  2. Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

    PubMed

    Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Gago, Gabriela; Spina, Lucie; Bardou, Fabienne; Lemassu, Anne; Quémard, Annaïk; Gramajo, Hugo

    2017-02-21

    Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases (ACCase) whose subunit composition and physiological roles have not yet been clearly established. A rather controversial data in the literature refers to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs; one of the substrates involved in the last step of condensation mediated by the polyketide synthase Pks13 to synthesize mature mycolic acids. Here we have successfully reconstituted the so called long-chain acyl-CoA carboxylase complex (LCC) from its purified components: the α-subunit AccA3, the ε-subunit AccE5 and the two β-subunits AccD4 and AccD5, and demonstrated that the four subunits are essential for its LCC activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor of the AccD5 subunit, that its role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen. This article is protected by copyright. All rights reserved.

  3. Regulation of renal fibrosis by Smad3 Thr388 phosphorylation.

    PubMed

    Qu, Xinli; Li, Xueling; Zheng, Yaowu; Ren, Yi; Puelles, Victor G; Caruana, Georgina; Nikolic-Paterson, David J; Li, Jinhua

    2014-04-01

    Transforming growth factor-β (TGF-β) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-β/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease.

  4. Modulation of soluble guanylate cyclase activity by phosphorylation.

    PubMed

    Murthy, Karnam S

    2004-11-01

    The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.

  5. Phosphoryl functionalized mesoporous silica for uranium adsorption

    NASA Astrophysics Data System (ADS)

    Xue, Guo; Yurun, Feng; Li, Ma; Dezhi, Gao; Jie, Jing; Jincheng, Yu; Haibin, Sun; Hongyu, Gong; Yujun, Zhang

    2017-04-01

    Phosphoryl functionalized mesoporous silica (TBP-SBA-15) was synthesized by modified mesoporous silica with γ-amino propyl triethoxy silane and tributyl phosphate. The obtained samples were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), small angle X-ray diffraction (SAXRD), thermo-gravimetric/differential thermalanalyzer (TG/DTA), N2 adsorption-desorption (BET) and Fourier transform infrared spectroscopy (FT-IR) techniques. Results showed that TBP-SBA-15 had large surface areas with ordered channel structure. Moreover, the effects of adsorption time, sorbent dose, solution pH, initial uranium concentration and temperature on the uranium adsorption behaviors were investigated. TBP-SBA-15 showed a high uranium adsorption capacity in a broad range of pH values. The U(VI) adsorption rate of TBP-SBA-15 was fast and nearly achieved completion in 10 min with the sorbent dose of 1 g/L. The U(VI) adsorption of TBP-SBA-15 followed the pseudo-second-order kinetic model and Freundlich isotherm model, indicating that the process was belonged to chemical adsorption. Furthermore, the thermodynamic parameters (ΔG0, ΔH0 and ΔS0) confirmed that the adsorption process was endothermic and spontaneous.

  6. Biomarker analysis of the MITO2 phase III trial of first-line treatment in ovarian cancer: predictive value of DNA-PK and phosphorylated ACC

    PubMed Central

    Perrone, Francesco; Baldassarre, Gustavo; Indraccolo, Stefano; Signoriello, Simona; Chiappetta, Gennaro; Esposito, Franca; Ferrandina, Gabriella; Franco, Renato; Mezzanzanica, Delia; Sonego, Maura; Zulato, Elisabetta; Zannoni, Gian F.; Canzonieri, Vincenzo; Scambia, Giovanni; Sorio, Roberto; Savarese, Antonella; Breda, Enrico; Scollo, Paolo; Ferro, Antonella; Tamberi, Stefano; Febbraro, Antonio; Natale, Donato; Maio, Massimo Di; Califano, Daniela; Scognamiglio, Giosuè; Lorusso, Domenica; Canevari, Silvana; Losito, Simona; Gallo, Ciro; Pignata, Sandro

    2016-01-01

    Background No biomarker is available to predict prognosis of patients with advanced ovarian cancer (AOC) and guide the choice of chemotherapy. We performed a prospective-retrospective biomarker study within the MITO2 trial on the treatment of AOC. Patients and methods: MITO2 is a randomised multicentre phase 3 trial conducted with 820 AOC patients assigned carboplatin/paclitaxel (carboplatin: AUC5, paclitaxel: 175 mg/m², every 3 weeks for 6 cycles) or carboplatin/PLD-pegylated liposomal doxorubicin (carboplatin: AUC5, PLD: 30 mg/m², every 3 weeks for 6 cycles) as first line treatment. Sixteen biomarkers (pathways of adhesion/invasion, apoptosis, transcription regulation, metabolism, and DNA repair) were studied in 229 patients, in a tissue microarray. Progression-free and overall survival were analysed with multivariable Cox model. Results After 72 months median follow-up, 594 progressions and 426 deaths were reported; there was no significant difference between the two arms in the whole trial. No biomarker had significant prognostic value. Statistically significant interactions with treatment were found for DNA-dependent protein kinase (DNA-PK) and phosphorylated acetyl-coenzymeA carboxylase (pACC), both predicting worse outcome for patients receiving carboplatin/paclitaxel. Conclusion These data show that in presence of DNA-PK or pACC overexpression, carboplatin/paclitaxel might be less effective than carboplatin/PLD as first line treatment of ovarian cancer patients. Further validation of these findings is warranted. PMID:27655643

  7. Catalytic roles of flexible regions at the active site of ribulose-bisphosphate carboxylase/oxygenase (Rubisco)

    SciTech Connect

    Hartman, F.C.; Harpel, M.R.; Chen, Yuh-Ru; Larson, E.M.; Larimer, F.W.

    1995-12-31

    Chemical and mutagenesis studies of Rubisco have identified Lys329 and Glu48 as active-site residues that are located in distinct, interacting domains from adjacent subunits. Crystallographic analyses have shown that Lys329 is the apical residue in a 12-residue flexible loop (loop 6) of the {Beta},{alpha}-barrel domain of the active site and that Glu48 resides at the end of helix B of the N-terminal domain of the active site. When phosphorylated ligands are bound by the enzyme, loop 6 adopts a closed conformation and, in concert with repositioning of helix B, thereby occludes the active site from the external environment. In this closed conformation, the {gamma}-carboxylate of Glu48 and the {epsilon}-amino group of Lys329 engage in intersubunit electrostatic interaction. By use of appropriate site-directed mutants of Rhodospirillum rubrum Rubisco, we are addressing several issues: the catalytic roles of Lys329 and Glu48, the functional significance of the intersubunit salt bridge comprised of these two residues, and the roles of loop 6 and helix B in stabilizing labile reaction intermediates. Characterization of novel products derived from misprocessing of D-ribulose-1,5-bisphosphate (RuBP) by the mutant proteins have illuminated the structure of the key intermediate in the normal oxygenase pathway.

  8. Comprehensive Analysis of Phosphorylated Proteins of E. coli Ribosomes

    PubMed Central

    Soung, George Y.; Miller, Jennifer L.; Koc, Hasan; Koc, Emine C.

    2009-01-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in twenty-four E. coli ribosomal proteins by tandem mass spectrometry. Specific detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining and antibodies for phospho-Ser, Thr, and Tyr, or by mass spectrometry equipped with a capability to detect addition and the loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given site of the phosphorylation in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins. PMID:19469554

  9. Signal processing by protein tyrosine phosphorylation in plants

    PubMed Central

    2011-01-01

    Protein phosphorylation is a reversible post-translational modification controlling many biological processes. Most phosphorylation occurs on serine and threonine, and to a less extend on tyrosine (Tyr). In animals, Tyr phosphorylation is crucial for the regulation of many responses such as growth or differentiation. Only recently with the development of mass spectrometry, it has been reported that Tyr phosphorylation is as important in plants as in animals. The genes encoding protein Tyr kinases and protein Tyr phosphatases have been identified in the Arabidopsis thaliana genome. Putative substrates of these enzymes, and thus Tyr-phosphorylated proteins have been reported by proteomic studies based on accurate mass spectrometry analysis of the phosphopeptides and phosphoproteins. Biochemical approaches, pharmacology and genetic manipulations have indicated that responses to stress and developmental processes involve changes in protein Tyr phosphorylation. The aim of this review is to present an update on Tyr phosphorylation in plants in order to better assess the role of this post-translational modification in plant physiology. PMID:21628997

  10. Phosphorylation of the pyruvate dehydrogenase complex isolated from Ascaris suum

    SciTech Connect

    Thissen, J.; Komuniecki, R.

    1987-05-01

    The pyruvate dehydrogenase complex (PDC) from body wall muscle of the porcine nematode, Ascaris suum, plays a pivotal role in anaerobic mitochondrial metabolism. As in mammalian mitochondria, PDC activity is inhibited by the phosphorylation of the ..cap alpha..PDH subunit, catalyzed by an associated PDH/sub a/ kinase. However, in contrast to PDC's isolated from all other eukaryotic sources, phosphorylation decreases the mobility of the ..cap alpha..PDH subunit on SDS-PAGE and permits the separation of the phosphorylated and nonphosphorylated ..cap alpha..PDH's. Phosphorylation and the inactivation of the Ascaris PDC correspond directly, and the additional phosphorylation that occurs after complete inactivation in mammalian PDC's is not observed. The purified ascarid PDC incorporates 10 nmoles /sup 32/P/mg P. Autoradiography of the radiolabeled PDC separated by SDS-PAGE yields a band which corresponds to the phosphorylated ..cap alpha..PDH and a second, faint band which is present only during the first three minutes of PDC inactivation, intermediate between the phosphorylated and nonphosphorylated ..cap alpha..PDH subunit. Tryptic digests of the /sup 32/P-PDC yields one major phosphopeptide, when separated by HPLC, and its amino acid sequence currently is being determined.

  11. Phosphorylation at serine 331 is required for Aurora B activation

    PubMed Central

    Petsalaki, Eleni; Akoumianaki, Tonia; Black, Elizabeth J.; Gillespie, David A.F.

    2011-01-01

    Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that phosphorylated Aurora B localizes to kinetochores in prometaphase cells. Chk1 kinase is essential for Ser331 phosphorylation during unperturbed prometaphase or during spindle disruption by taxol but not nocodazole. Phosphorylation at Ser331 is required for optimal phosphorylation of INCENP at TSS residues, for Survivin association with the chromosomal passenger complex, and for complete Aurora B activation, but it is dispensable for Aurora B localization to centromeres, for autophosphorylation at threonine 232, and for association with INCENP. Overexpression of Aurora BS331A, in which Ser331 is mutated to alanine, results in spontaneous chromosome missegregation, cell multinucleation, unstable binding of BubR1 to kinetochores, and impaired mitotic delay in the presence of taxol. We propose that Chk1 phosphorylates Aurora B at Ser331 to fully induce Aurora B kinase activity. These results indicate that phosphorylation at Ser331 is an essential mechanism for Aurora B activation. PMID:22024163

  12. Role of Phosphorylated HDAC4 in Stroke-Induced Angiogenesis

    PubMed Central

    Liu, Juan; Zhou, Xiang; Li, Qing; Zhou, Shu-Min; Hu, Bin; Hu, Guo-Wen; Niu, Xin; Guo, Shang-Chun

    2017-01-01

    Acetylation or deacetylation of chromatin proteins and transcription factors is part of a complex signaling system that is involved in the control of neurological disorders. Recent studies have demonstrated that histone deacetylases (HDACs) exert protective effects in attenuating neuronal injury after ischemic insults. Class IIa HDAC4 is highly expressed in the brain, and neuronal activity depends on the nucleocytoplasmic shuttling of HDAC4. However, little is known about HDAC4 and its roles in ischemic stroke. In this study, we report that phosphorylation of HDAC4 was remarkably upregulated after stroke and blockade of HDAC4 phosphorylation with GÖ6976 repressed stroke-induced angiogenesis. Phosphorylation of HDAC4 was also increased in endothelial cells hypoxia model and suppression of HDAC4 phosphorylation inhibited the tube formation and migration of endothelial cells in vitro. Furthermore, in addition to the inhibition of angiogenesis, blockade of HDAC4 phosphorylation suppressed the expression of genes downstream of HIF-VEGF signaling in vitro and in vivo. These data indicate that phosphorylated HDAC4 may serve as an important regulator in stroke-induced angiogenesis. The protective mechanism of phosphorylated HDAC4 is associated with HIF-VEGF signaling, implicating a novel therapeutic target in stroke. PMID:28127553

  13. A phosphorylation map of the bovine papillomavirus E1 helicase

    PubMed Central

    Lentz, Michael R; Stevens, Stanley M; Raynes, Joshua; Elkhoury, Nancy

    2006-01-01

    Background Papillomaviruses undergo a complex life cycle requiring regulated DNA replication. The papillomavirus E1 helicase is essential for viral DNA replication and plays a key role in controlling viral genome copy number. The E1 helicase is regulated at least in part by protein phosphorylation, however no systematic approach to phosphate site mapping has been attempted. We have utilized mass spectrometry of purified bovine papillomavirus E1 protein to identify and characterize new sites of phosphorylation. Results Mass spectrometry and in silico sequence analysis were used to identify phosphate sites on the BPV E1 protein and kinases that may recognize these sites. Five new and two previously known phosphorylation sites were identified. A phosphate site map was created and used to develop a general model for the role of phosphorylation in E1 function. Conclusion Mass spectrometric analysis identified seven phosphorylated amino acids on the BPV E1 protein. Taken with three previously identified sites, there are at least ten phosphoamino acids on BPV E1. A number of kinases were identified by sequence analysis that could potentially phosphorylate E1 at the identified positions. Several of these kinases have known roles in regulating cell cycle progression. A BPV E1 phosphate map and a discussion of the possible role of phosphorylation in E1 function are presented. PMID:16524476

  14. Structural Impact of Tau Phosphorylation at Threonine 231.

    PubMed

    Schwalbe, Martin; Kadavath, Harindranath; Biernat, Jacek; Ozenne, Valery; Blackledge, Martin; Mandelkow, Eckhard; Zweckstetter, Markus

    2015-08-04

    Phosphorylation of the microtubule-associated protein Tau influences the assembly and stabilization of microtubules and is deregulated in several neurodegenerative diseases. The high flexibility of Tau, however, has prevented an atomic-level description of its phosphorylation-induced structural changes. Employing an extensive set of distance and orientational restraints together with a novel ensemble calculation approach, we determined conformational ensembles of Tau fragments in the non-phosphorylated state and, when phosphorylated at T231/S235 or T231/S235/S237/S238, four important sites of phosphorylation in Alzheimer disease. Comparison of the molecular ensembles showed that phosphorylation of the regulatory T231 does not perturb the backbone conformation of the proximal microtubule-binding (225)KVAVVR(230) motif. Instead, phosphorylated T231 selectively engages in a salt bridge with R230 that can compete with the formation of intermolecular salt bridges to tubulin. Our study provides an ensemble description which will be useful for the analysis of conformational transitions in Tau and other intrinsically disordered proteins.

  15. MSK1 activity is controlled by multiple phosphorylation sites

    PubMed Central

    McCOY, Claire E.; Campbell, David G.; Deak, Maria; Bloomberg, Graham B.; Arthur, J. Simon C.

    2004-01-01

    MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2. PMID:15568999

  16. Cyanogen induced phosphorylation of D-fructose. [prebiotic modeling

    NASA Technical Reports Server (NTRS)

    Degani, CH.; Kawatsuji, M.; Halmann, M.

    1975-01-01

    It has been demonstrated that a phosphorylated sugar, identified as alpha-D-fructopyranose, can be formed as the result of cyanogen-induced phosphorylation of D-fructose at pH 8.8. The product was isolated from barium and cyclohexylammonium salts and identified on the basis of its chromatographic and electrophoretic properties, its lability to hydrolysis by alkaline phosphatase, the rate of its acid-catalyzed hydrolysis, and the results of periodate oxidation and optical rotatory measurements. These results support the suggestion that the cyanogen-induced phosphorylation of free sugars could be a possible process for formation of sugar phosphates under prebiotic conditions (Halman et al., 1969).

  17. Rosamines Targeting the Cancer Oxidative Phosphorylation Pathway

    PubMed Central

    Lim, Siang Hui; Wu, Liangxing; Kiew, Lik Voon; Chung, Lip Yong; Burgess, Kevin; Lee, Hong Boon

    2014-01-01

    Reprogramming of energy metabolism is pivotal to cancer, so mitochondria are potential targets for anticancer therapy. A prior study has demonstrated the anti-proliferative activity of a new class of mitochondria-targeting rosamines. This present study describes in vitro cytotoxicity of second-generation rosamine analogs, their mode of action, and their in vivo efficacies in a tumor allografted mouse model. Here, we showed that these compounds exhibited potent cytotoxicity (average IC50<0.5 µM), inhibited Complex II and ATP synthase activities of the mitochondrial oxidative phosphorylation pathway and induced loss of mitochondrial transmembrane potential. A NCI-60 cell lines screen further indicated that rosamine analogs 4 and 5 exhibited potent antiproliferative effects with Log10GI50 = −7 (GI50 = 0.1 µM) and were more effective against a colorectal cancer sub-panel than other cell lines. Preliminary in vivo studies on 4T1 murine breast cancer-bearing female BALB/c mice indicated that treatment with analog 5 in a single dosing of 5 mg/kg or a schedule dosing of 3 mg/kg once every 2 days for 6 times (q2d×6) exhibited only minimal induction of tumor growth delay. Our results suggest that rosamine analogs may be further developed as mitochondrial targeting agents. Without a doubt proper strategies need to be devised to enhance tumor uptake of rosamines, i.e. by integration to carrier molecules for better therapeutic outcome. PMID:24622277

  18. Role of energy in oxidative phosphorylation.

    PubMed

    Matsuno-Yagi, A; Hatefi, Y

    1988-08-01

    This article reviews the current status of information regarding the role of energy in the process of oxidative phosphorylation by mitochondria. The available data suggest that in submitochondrial particles (SMP) energy is utilized for the binding of ADP and Pi and for the release of ATP bound at the catalytic sites of F1-ATPase. The process of ATP synthesis on the surface of F1 from F1-bound ADP and Pi appears to be associated with negligible free energy change. The rate of energy production by the respiratory chain modulates the kinetics of ATP synthesis between a low Km (for ADP and Pi)-low Vmax mode and a high Km-high Vmax mode. The Km extremes for ADP are 2-3 microM and 120-150 microM, and Vmax for ATP synthesis at high rates of energy production by bovine-heart SMP is about 440 S-1 (mole F1)-1 at 30 degrees C, which corresponds to 11 mumol ATP (min.mg of protein)-1. The interaction of dicyclohexylcarbodiimide (DCCD) or oligomycin at the proteolipid (subunit c) of the membrane sector (F0) of the ATP synthase complex alters the mode of ATP binding at the catalytic sites of F1, probably to one of lower affinity. It has been suggested that protonic energy might be conveyed to the catalytic sites of F1 in an analogous manner, i.e., via conformation changes in the ATP synthase complex initiated by proton-induced alterations in the structure of the DCCD-binding proteolipid. Finally, the relationship between the steady-state membrane potential (delta psi) and the rates of electron transfer and ATP synthesis has been discussed. It has been shown, in agreement with the delocalized chemiosmotic mechanism, that under appropriate conditions delta psi is exquisitely sensitive to changes in the rates of energy production and consumption.

  19. Mass spectrometric phosphoproteome analysis of HIV-infected brain reveals novel phosphorylation sites and differential phosphorylation patterns

    PubMed Central

    Uzasci, Lerna; Auh, Sungyoung; Cotter, Robert J.; Nath, Avindra

    2016-01-01

    Purpose To map the phosphoproteome and identify changes in the phosphorylation patterns in the HIV-infected and uninfected brain using high-resolution mass spectrometry. Experimental Design Parietal cortex from brain of individuals with and without HIV infection were lysed and trypsinized. The peptides were labeled with iTRAQ reagents, combined, phospho-enriched by titanium dioxide chromatography, and analyzed by LC-MS/MS with high-resolution. Results Our phosphoproteomic workflow resulted in the identification of 112 phosphorylated proteins and 17 novel phosphorylation sites in all the samples that were analyzed. The phosphopeptide sequences were searched for kinase substrate motifs which revealed potential kinases involved in important signaling pathways. The site-specific phosphopeptide quantification showed that peptides from neurofilament medium polypeptide, myelin basic protein, and 2′–3′-cyclic nucleotide-3′ phosphodiesterase have relatively higher phosphorylation levels during HIV infection. Clinical Relevance This study has enriched the global phosphoproteome knowledge of the human brain by detecting novel phosphorylation sites on neuronal proteins and identifying differentially phosphorylated brain proteins during HIV infection. Kinases that lead to unusual phosphorylations could be therapeutic targets for the treatment of HIV-associated neurocognitive disorders (HAND). PMID:26033855

  20. Calcineurin regulates phosphorylation status of transcription factor osterix.

    PubMed

    Okamura, Hirohiko; Amorim, Bruna Rabelo; Wang, Jie; Yoshida, Kaya; Haneji, Tatsuji

    2009-02-06

    Osterix is an osteoblast-specific transcriptional factor that is essential for osteoblast differentiation and bone formation. Calcineurin regulates bone formation through modulating osteoblast differentiation. However, post-translational modification of osterix such as phosphorylation and interactions between osterix and calcineurin remains unclear. In the present study, we demonstrated that calcineurin interacted with osterix determined by immunoprecipitation assay and Western analysis. Immunocytochemical study also revealed that osterix and calcineurin were co-localized in nucleus. Deletion of calcineurin binding motif on osterix molecule disrupted osterix-calcineurin interaction. Phosphorylation status of osterix was augmented by treatment with phosphatase inhibitors, FK506 and calyculin A. In contrast, treatment of recombinant calcineurin reduced phosphorylation status of osterix. Our present study suggests that calcineurin has an important role in the function of osterix through its modification of phosphorylation.

  1. Methods for generating phosphorylation site-specific immunological reagents

    DOEpatents

    Anderson, Carl W.; Appella, Ettore; Sakaguchi, Kazuyasu

    2001-01-01

    The present invention provides methods for generating phosphorylation site-specific immunological reagents. More specifically, a phosphopeptide mimetic is incorporated into a polypeptide in place of a phosphorylated amino acid. The polypeptide is used as antigen by standard methods to generate either monoclonal or polyclonal antibodies which cross-react with the naturally phosphorylated polypeptide. The phosphopeptide mimetic preferably contains a non-hydrolyzable linkage from the appropriate carbon atom of the amino acid residue to a phosphate group. A preferred linkage is a CF.sub.2 group. Such a linkage is used to generate the phosphoserine mimetic F.sub.2 Pab, which is incorporated into a polypeptide sequence derived from p53 to produce antibodies which recognize a specific phosphorylation state of p53. A CF.sub.2 group linkage is also used to produce the phosphothreonine mimetic F.sub.2 Pmb, and to produce the phosphotyrosine mimetic, F.sub.2 Pmp.

  2. On Stationary States in the Double Phosphorylation-dephosphorylation Cycle

    NASA Astrophysics Data System (ADS)

    Bersani, Alberto Maria; Dell'Acqua, Guido; Tomassetti, Giovanna

    2011-09-01

    In this paper we study the double phosphorylation-dephosphorylation cycle, which is a special case of multiple futile cycle. We study the stationary states, finding some classes of explicit solutions.

  3. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    NASA Astrophysics Data System (ADS)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  4. Aging effects on oxidative phosphorylation in rat adrenocortical mitochondria.

    PubMed

    Solinas, Paola; Fujioka, Hisashi; Radivoyevitch, Tomas; Tandler, Bernard; Hoppel, Charles L

    2014-06-01

    Does aging in itself lead to alteration in adrenocortical mitochondrial oxidative phosphorylation? Mitochondria from Fischer 344 (F344) rats (6 and 24 months old), Brown Norway rats (6 and 32 months old) and F344-Brown Norway hybrid rats (6 and 30 months old) were compared. Mitochondria were isolated from extirpated adrenal cortex. The yields of mitochondria were quantitatively similar in all rat strains irrespective of age. In order to assess the activity of each mitochondrial complex, several different substrates were tested and the rate of oxidative phosphorylation measured. Aging does not affect mitochondrial activity except in the F344 rat adrenal cortex where the maximal ADP-stimulated oxidative phosphorylation decreased with age. We hypothesize that impaired synthesis of steroid hormones by the adrenal cortex with age in F344 rats might be due to decreased adrenocortical mitochondrial oxidative phosphorylation. We conclude that aging results in adrenocortical mitochondria effects that are non-uniform across different rat strains.

  5. Phosphorylation of Mad controls competition between wingless and BMP signaling.

    PubMed

    Eivers, Edward; Demagny, Hadrien; Choi, Renee H; De Robertis, Edward M

    2011-10-11

    Bone morphogenetic proteins (BMPs) and Wnts are growth factors that provide essential patterning signals for cell proliferation and differentiation. Here, we describe a molecular mechanism by which the phosphorylation state of the Drosophila transcription factor Mad determines its ability to transduce either BMP or Wingless (Wg) signals. Previously, Mad was thought to function in gene transcription only when phosphorylated by BMP receptors. We found that the unphosphorylated form of Mad was required for canonical Wg signaling by interacting with the Pangolin-Armadillo transcriptional complex. Phosphorylation of the carboxyl terminus of Mad by BMP receptor directed Mad toward BMP signaling, thereby preventing Mad from functioning in the Wg pathway. The results show that Mad has distinct signal transduction roles in the BMP and Wnt pathways depending on its phosphorylation state.

  6. Biological phosphoryl-transfer reactions: understanding mechanism and catalysis.

    PubMed

    Lassila, Jonathan K; Zalatan, Jesse G; Herschlag, Daniel

    2011-01-01

    Phosphoryl-transfer reactions are central to biology. These reactions also have some of the slowest nonenzymatic rates and thus require enormous rate accelerations from biological catalysts. Despite the central importance of phosphoryl transfer and the fascinating catalytic challenges it presents, substantial confusion persists about the properties of these reactions. This confusion exists despite decades of research on the chemical mechanisms underlying these reactions. Here we review phosphoryl-transfer reactions with the goal of providing the reader with the conceptual and experimental background to understand this body of work, to evaluate new results and proposals, and to apply this understanding to enzymes. We describe likely resolutions to some controversies, while emphasizing the limits of our current approaches and understanding. We apply this understanding to enzyme-catalyzed phosphoryl transfer and provide illustrative examples of how this mechanistic background can guide and deepen our understanding of enzymes and their mechanisms of action. Finally, we present important future challenges for this field.

  7. Biological Phosphoryl-Transfer Reactions: Understanding Mechanism and Catalysis

    PubMed Central

    Lassila, Jonathan K.; Zalatan, Jesse G.; Herschlag, Daniel

    2012-01-01

    Phosphoryl-transfer reactions are central to biology. These reactions also have some of the slowest nonenzymatic rates and thus require enormous rate accelerations from biological catalysts. Despite the central importance of phosphoryl transfer and the fascinating catalytic challenges it presents, substantial confusion persists about the properties of these reactions. This confusion exists despite decades of research on the chemical mechanisms underlying these reactions. Here we review phosphoryl-transfer reactions with the goal of providing the reader with the conceptual and experimental background to understand this body of work, to evaluate new results and proposals, and to apply this understanding to enzymes. We describe likely resolutions to some controversies, while emphasizing the limits of our current approaches and understanding. We apply this understanding to enzyme-catalyzed phosphoryl transfer and provide illustrative examples of how this mechanistic background can guide and deepen our understanding of enzymes and their mechanisms of action. Finally, we present important future challenges for this field. PMID:21513457

  8. Enrichment of phosphorylated peptides and proteins by selective precipitation methods.

    PubMed

    Rainer, Matthias; Bonn, Günther K

    2015-01-01

    Protein phosphorylation is one of the most prominent post-translational modifications involved in the regulation of cellular processes. Fundamental understanding of biological processes requires appropriate bioanalytical methods for selectively enriching phosphorylated peptides and proteins. Most of the commonly applied enrichment approaches include chromatographic materials including Fe(3+)-immobilized metal-ion affinity chromatography or metal oxides. In the last years, the introduction of several non-chromatographic isolation technologies has increasingly attracted the interest of many scientists. Such approaches are based on the selective precipitation of phosphorylated peptides and proteins by applying various metal cations. The excellent performance of precipitation-based enrichment methods can be explained by the absence of any stationary phase, resin or sorbent, which usually leads to unspecific binding. This review provides an overview of recently published methods for the selective precipitation of phosphorylated peptides and proteins.

  9. Doubling down on phosphorylation as a variable peptide modification.

    PubMed

    Cooper, Bret

    2016-09-01

    Some mass spectrometrists believe that searching for variable PTMs like phosphorylation of serine or threonine when using database-search algorithms to interpret peptide tandem mass spectra will increase false-positive matching. The basis for this is the premise that the algorithm compares a spectrum to both a nonphosphorylated peptide candidate and a phosphorylated candidate, which is double the number of candidates compared to a search with no possible phosphorylation. Hence, if the search space doubles, false-positive matching could increase accordingly as the algorithm considers more candidates to which false matches could be made. In this study, it is shown that the search for variable phosphoserine and phosphothreonine modifications does not always double the search space or unduly impinge upon the FDR. A breakdown of how one popular database-search algorithm deals with variable phosphorylation is presented.

  10. 3,5 Diiodo-L-Thyronine (T2) Does Not Prevent Hepatic Steatosis or Insulin Resistance in Fat-Fed Sprague Dawley Rats

    PubMed Central

    Vatner, Daniel F.; Snikeris, Jaclyn; Popov, Violeta; Perry, Rachel J.; Rahimi, Yasmeen; Samuel, Varman T.

    2015-01-01

    Thyroid hormone mimetics are alluring potential therapies for diseases like dyslipidemia, nonalcoholic fatty liver disease (NAFLD), and insulin resistance. Though diiodothyronines are thought inactive, pharmacologic treatment with 3,5- Diiodo-L-Thyronine (T2) reportedly reduces hepatic lipid content and improves glucose tolerance in fat-fed male rats. To test this, male Sprague Dawley rats fed a safflower-oil based high-fat diet were treated with T2 (0.25 mg/kg-d) or vehicle. Neither 10 nor 30 days of T2 treatment had an effect on weight, adiposity, plasma fatty acids, or hepatic steatosis. Insulin action was quantified in vivo by a hyperinsulinemic-euglycemic clamp. T2 did not alter fasting plasma glucose or insulin concentration. Basal endogenous glucose production (EGP) rate was unchanged. During the clamp, there was no difference in insulin stimulated whole body glucose disposal. Insulin suppressed EGP by 60% ± 10 in T2-treated rats as compared with 47% ± 4 suppression in the vehicle group (p = 0.32). This was associated with an improvement in hepatic insulin signaling; insulin stimulated Akt phosphorylation was ~2.5 fold greater in the T2-treated group as compared with the vehicle-treated group (p = 0.003). There was no change in expression of genes thought to mediate the effect of T2 on hepatic metabolism, including genes that regulate hepatic lipid oxidation (ppara, carnitine palmitoyltransferase 1a), genes that regulate hepatic fatty acid synthesis (srebp1c, acetyl coa carboxylase, fatty acid synthase), and genes involved in glycolysis and gluconeogenesis (L-pyruvate kinase, glucose 6 phosphatase). Therefore, in contrast with previous reports, in Sprague Dawley rats fed an unsaturated fat diet, T2 administration failed to improve NAFLD or whole body insulin sensitivity. Though there was a modest improvement in hepatic insulin signaling, this was not associated with significant differences in hepatic insulin action. Further study will be necessary before

  11. Phosphorylation sites in human erythrocyte band 3 protein.

    PubMed

    Yannoukakos, D; Vasseur, C; Piau, J P; Wajcman, H; Bursaux, E

    1991-01-30

    The human red cell anion-exchanger, band 3 protein, is one of the main phosphorylated proteins of the erythrocyte membrane. Previous studies from this laboratory have shown that ATP-depletion of the red blood cell decreased the anion-exchange rate, suggesting that band 3 protein phosphorylation could be involved in the regulation of anion transport function (Bursaux et al. (1984) Biochim. Biophys. Acta 777, 253-260). Phosphorylation occurs mainly on the cytoplasmic domain of the protein and the major site of phosphorylation was assigned to tyrosine-8 (Dekowski et al. (1983) J. Biol. Chem. 258, 2750-2753). This site being very far from the integral, anion-exchanger domain, the aim of the present study was to determine whether phosphorylation sites exist in the integral domain. The phosphorylation reaction was carried out on isolated membranes in the presence of [gamma-32P]ATP and phosphorylated band 3 protein was then isolated. Both the cytoplasmic and the membrane spanning domains were purified. The predominant phosphorylation sites were found on the cytoplasmic domain. RP-HPLC analyses of the tryptic peptides of whole band 3 protein, and of the isolated cytoplasmic and membrane-spanning domains allowed for the precise localization of the phosphorylated residues. 80% of the label was found in the N-terminal tryptic peptide (T-1), (residues 1-56). In this region, all the residues susceptible to phosphorylation were labeled but in varying proportion. Under our conditions, the most active membrane kinase was a tyrosine kinase, activated preferentially by Mn2+ but also by Mg2+. Tyrosine-8 was the main phosphate acceptor residue (50-70%) of the protein, tyrosine-21 and tyrosine-46 residues were also phosphorylated but to a much lesser extent. The main targets of membrane casein kinase, preferentially activated by Mg2+, were serine-29, serine-50, and threonine(s)-39, -42, -44, -48, -49, -54 residue(s) located in the T-1 peptide. A tyrosine phosphatase activity was

  12. Tau Phosphorylation by GSK3 in Different Conditions

    PubMed Central

    Avila, Jesús; León-Espinosa, Gonzalo; García, Esther; García-Escudero, Vega; Hernández, Félix; DeFelipe, Javier

    2012-01-01

    Almost a 20% of the residues of tau protein are phosphorylatable amino acids: serine, threonine, and tyrosine. In this paper we comment on the consequences for tau of being a phosphoprotein. We will focus on serine/threonine phosphorylation. It will be discussed that, depending on the modified residue in tau molecule, phosphorylation could be protective, in processes like hibernation, or toxic like in development of those diseases known as tauopathies, which are characterized by an hyperphosphorylation and aggregation of tau. PMID:22675648

  13. Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels.

    PubMed

    Tian, Lijun; Jeffries, Owen; McClafferty, Heather; Molyvdas, Adam; Rowe, Iain C M; Saleem, Fozia; Chen, Lie; Greaves, Jennifer; Chamberlain, Luke H; Knaus, Hans-Guenther; Ruth, Peter; Shipston, Michael J

    2008-12-30

    Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming alpha-subunits. However, the molecular mechanism(s) by which phosphorylation of the alpha-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single alpha-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.

  14. Phosphorylation of proteins during human myometrial contractions: A phosphoproteomic approach.

    PubMed

    Hudson, Claire A; López Bernal, Andrés

    2017-01-22

    Phasic myometrial contractility is a key component of human parturition and the contractions are driven by reversible phosphorylation of myosin light chains catalyzed by the calcium (Ca(2+))-dependent enzyme myosin light chain kinase (MYLK). Other yet unknown phosphorylation or de-phosphorylation events may contribute to myometrial contraction and relaxation. In this study we have performed a global phosphoproteomic analysis of human myometrial tissue using tandem mass tagging to detect changes in the phosphorylation status of individual myometrial proteins during spontaneous and oxytocin-driven phasic contractions. We were able to detect 22 individual phosphopeptides whose relative ratio changed (fold > 2 or < 0.5) in response to spontaneous or oxytocin-stimulated contraction. The most significant changes in phosphorylation were to MYLK on serine 1760, a site associated with reductions in calmodulin binding and subsequent kinase activity. Phosphorylated MYLK (ser1760) increased significantly during spontaneous (9.83 ± 3.27 fold, P < 0.05) and oxytocin -induced (18.56 ± 8.18 fold, P < 0.01) contractions and we were able to validate these data using immunoblotting. Pathway analysis suggested additional proteins involved in calcium signalling, cGMP-PRKG signalling, adrenergic signalling and oxytocin signalling were also phosphorylated during contractions. This study demonstrates that a global phosphoproteomic analysis of myometrial tissue is a sensitive approach to detect changes in the phosphorylation of proteins during myometrial contractions, and provides a platform for further validation of these changes and for identification of their functional significance.

  15. Phosphorylation of mammalian initiation factor eIF-4B

    SciTech Connect

    Duncan, R.F.; Milburn, S.C.; Cooper, R.; Gould, K.; Hunter, T.; Hershey, J.W.B.

    1987-05-01

    The phosphorylation of initiation factors appears to be an important mechanism for regulating the rate of translation in mammalian cells. eIF-4B (80 kDa) purified from HeLa cells exhibits a complex array of 8 to 12 spots when analyzed by 2-dimensional isoelectric focusing - SDS polyacrylamide gel electrophoresis. A similar array of eIF-4B spots is seen when total lysate proteins are analyzed by immunoblotting with anti-eIF-4B antiserum or with antibodies affinity-purified from the most basic eIF-4B spot. The multiple forms of eIF-4B are due to phosphorylation, since all but the most basic spot are labeled with (/sup 32/P)phosphate in vivo and the action of alkaline phosphatase in vitro reduces the array to only two spots. Tryptic peptide maps of phosphopeptides from each of the various isoelectric variants of eIF-4B show a similar complexity, suggesting that a number of different sites are phosphorylated in a random order. When serum-deprived HeLa cells are treated with phorbol ester, both the protein synthesis rate and the extent of eIF-4B phosphorylation increase, suggesting that C kinase may be a regulator of translation. Purified C kinase phosphorylates a number of pure initiation factors in vitro, but eIF-4B is the strongest target protein. When pure eIF-4B is treated, the entire mass of eIF-4B is shifted to the most acidic spots, indicating very strong phosphorylation. Attempts are being made to detect differences in the in vitro activities of the non-phosphorylated and highly phosphorylated forms.

  16. Protein kinase C alpha-dependent phosphorylation of Golgi proteins.

    PubMed

    Radau, B; Otto, A; Müller, E C; Westermann, P

    2000-07-01

    Golgi-enriched membranes were phosphorylated in order to understand the mechanism for protein kinase C (PKC) regulation of exocytic vesicle formation at the trans-Golgi network. Two of the main PKC substrates were identified as MARCKS and Mac-MARCKS by two-dimensional electrophoresis (2-DE) and mass spectrometric sequencing. Annexin IV and profilin I, two other Golgi-associated proteins--although known as in vitro PKC substrates--were not phosphorylated in the Golgi-bound state.

  17. Protein phosphorylation and its role in archaeal signal transduction

    PubMed Central

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C.; Albers, Sonja-Verena; Siebers, Bettina

    2016-01-01

    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

  18. Plasma membrane ATPase of red beet forms a phosphorylated intermediate.

    PubMed

    Briskin, D P; Poole, R J

    1983-03-01

    When a plasma membrane-enriched fraction isolated from red beet (Beta vulgaris L.) was incubated in the presence of 40 micromolar [gamma-(32)P] ATP, 40 micromolar MgSO(4) at pH 6.5, a rapidly turning over phosphorylated protein was formed. Phosphorylation of the protein was substrate-specific for ATP, sensitive to diethylstilbestrol and vanadate, but insensitive to azide. When the dephosphorylation reaction was specifically studied, KCl was found to increase the turnover of the phosphorylated protein consistent with its stimulatory effect upon plasma membrane ATPase. The protein-bound phosphate was found to be most stable at a pH between 2 and 3 and under cold temperature, suggesting that the protein phosphate bond was an acyl-phosphate. When the phosphorylated protein was analyzed with lithium dodecyl sulfate gel electrophoresis, a labeled polypeptide with a molecular weight of about 100,000 daltons was observed. Phosphorylation of this polypeptide was rapidly turning over and Mg-dependent. It is concluded that the phosphorylation observed represents a reaction intermediate of the red beet plasma membrane ATPase.

  19. Formation and Dissociation of Phosphorylated Peptide Radical Cations

    NASA Astrophysics Data System (ADS)

    Kong, Ricky P. W.; Quan, Quan; Hao, Qiang; Lai, Cheuk-Kuen; Siu, Chi-Kit; Chu, Ivan K.

    2012-12-01

    In this study, we generated phosphoserine- and phosphothreonine-containing peptide radical cations through low-energy collision-induced dissociation (CID) of the ternary metal-ligand phosphorylated peptide complexes [CuII(terpy) p M]·2+ and [CoIII(salen) p M]·+ [ p M: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N, N '-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations ( p M·+) revealed fascinating gas-phase radical chemistry, yielding (1) charge-directed b- and y-type product ions, (2) radical-driven product ions through cleavages of peptide backbones and side chains, and (3) different degrees of formation of [M - H3PO4]·+ species through phosphate ester bond cleavage. The CID spectra of the p M·+ species and their non-phosphorylated analogues featured fragment ions of similar sequence, suggesting that the phosphoryl group did not play a significant role in the fragmentation of the peptide backbone or side chain. The extent of neutral H3PO4 loss was influenced by the peptide sequence and the initial sites of the charge and radical. A preliminary density functional theory study, at the B3LYP 6-311++G(d,p) level of theory, of the neutral loss of H3PO4 from a prototypical model— N-acetylphosphorylserine methylamide—revealed several factors governing the elimination of neutral phosphoryl groups through charge- and radical-induced mechanisms.

  20. Identification of Phosphorylation Sites on Extracellular Corneal Epithelial Cell Maspin

    PubMed Central

    Narayan, Malathi; Mirza, Shama P.; Twining, Sally S.

    2011-01-01

    Maspin, a 42-kDa non classical serine protease inhibitor (serpin) is expressed by epithelial cells of various tissues including the cornea. The protein localizes to the nucleus and cytosol, and is present in the extracellular space. While extracellular maspin regulates corneal stromal fibroblast adhesion and inhibits angiogenesis during wound healing in the cornea, the molecular mechanism of its extracellular functions is unclear. We hypothesized that identifying post-translational modifications of maspin, such as phosphorylation, may help decipher its mode of action. The focus of this study was on the identification of phosphorylation sites on extracellular maspin, since the extracellular form of the molecule is implicated in several functions. Multi-stage fragmentation mass spectrometry was used to identify sites of phosphorylation on extracellular corneal epithelial cell maspin. A total of eight serine and threonine phosphorylation sites (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310, Ser316) were identified on the extracellular forms of the molecule. Phosphorylation of tyrosine residues on extracellular maspin was not detected on extracellular maspin from corneal epithelial cell, in contrast to breast epithelial cells. This study provides the basis for further investigation into the functional role of phosphorylation of corneal epithelial maspin. PMID:21365746

  1. Control of serotonin transporter phosphorylation by conformational state

    PubMed Central

    Zhang, Yuan-Wei; Turk, Benjamin E.

    2016-01-01

    Serotonin transporter (SERT) is responsible for reuptake and recycling of 5-hydroxytryptamine (5-HT; serotonin) after its exocytotic release during neurotransmission. Mutations in human SERT are associated with psychiatric disorders and autism. Some of these mutations affect the regulation of SERT activity by cGMP-dependent phosphorylation. Here we provide direct evidence that this phosphorylation occurs at Thr276, predicted to lie near the cytoplasmic end of transmembrane helix 5 (TM5). Using membranes from HeLa cells expressing SERT and intact rat basophilic leukemia cells, we show that agents such as Na+ and cocaine that stabilize outward-open conformations of SERT decreased phosphorylation and agents that stabilize inward-open conformations (e.g., 5-HT, ibogaine) increased phosphorylation. The opposing effects of the inhibitors cocaine and ibogaine were each reversed by an excess of the other inhibitor. Inhibition of phosphorylation by Na+ and stimulation by ibogaine occurred at concentrations that induced outward opening and inward opening, respectively, as measured by the accessibility of cysteine residues in the extracellular and cytoplasmic permeation pathways, respectively. The results are consistent with a mechanism of SERT regulation that is activated by the transport of 5-HT, which increases the level of inward-open SERT and may lead to unwinding of the TM5 helix to allow phosphorylation. PMID:27140629

  2. Phosphorescent sensor for phosphorylated peptides based on an iridium complex.

    PubMed

    Kang, Jung Hyun; Kim, Hee Jin; Kwon, Tae-Hyuk; Hong, Jong-In

    2014-07-03

    A bis[(4,6-difluorophenyl)pyridinato-N,C(2')]iridium(III) picolinate (FIrpic) derivative coupled with bis(Zn(2+)-dipicolylamine) (ZnDPA) was developed as a sensor (1) for phosphorylated peptides, which are related to many cellular mechanisms. As a control, a fluorescent sensor (2) based on anthracene coupled to ZnDPA was also prepared. When the total negative charge on the phosphorylated peptides was changed to -2, -4, and -6, the emission intensity of sensor 1 gradually increased by factors of up to 7, 11, and 16, respectively. In contrast, there was little change in the emission intensity of sensor 1 upon the addition of a neutral phosphorylated peptide, non-phosphorylated peptides, or various anions such as CO3(2-), NO3(-), SO4(2-), phosphate, azide, and pyrophosphate. Furthermore, sensor 1 could be used to visually discriminate between phosphorylated peptides and adenosine triphosphate in aqueous solution under a UV-vis lamp, unlike fluorescent sensor 2. This enhanced luminance of phosphorescent sensor 1 upon binding to a phosphorylated peptide is attributed to a reduction in the repulsion between the Zn(2+) ions due to the phenoxy anion, its strong metal-to-ligand charge transfer character, and a reduction in self-quenching.

  3. ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation

    PubMed Central

    Lievens, Patricia Marie-Jeanne; Kuznetsova, Tatiana; Kochlamazashvili, Gaga; Cesca, Fabrizia; Gorinski, Natalya; Galil, Dalia Abdel; Cherkas, Volodimir; Ronkina, Natalia; Lafera, Juri; Gaestel, Matthias

    2016-01-01

    The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis. PMID:27247265

  4. Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides

    PubMed Central

    Bertran-Vicente, Jordi; Penkert, Martin; Nieto-Garcia, Olaia; Jeckelmann, Jean-Marc; Schmieder, Peter; Krause, Eberhard; Hackenberger, Christian P. R.

    2016-01-01

    In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICBGlc, which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes. PMID:27586301

  5. The regulation of AMP-activated protein kinase by phosphorylation.

    PubMed Central

    Stein, S C; Woods, A; Jones, N A; Davison, M D; Carling, D

    2000-01-01

    The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr(172) on AMPK activity. Mutation of Thr(172) to an aspartic acid residue (T172D) in either alpha1 or alpha2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr(172) accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the alpha subunit and one site on the beta subunit. Furthermore, we provide evidence that phosphorylation of Thr(172) may be involved in the sensitivity of the AMPK complex to AMP. PMID:10642499

  6. Structural basis for Mep2 ammonium transceptor activation by phosphorylation

    PubMed Central

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C.

    2016-01-01

    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. PMID:27088325

  7. Synthetic phosphorylation of p38α recapitulates protein kinase activity.

    PubMed

    Chooi, K Phin; Galan, Sébastien R G; Raj, Ritu; McCullagh, James; Mohammed, Shabaz; Jones, Lyn H; Davis, Benjamin G

    2014-02-05

    Through a "tag-and-modify" protein chemical modification strategy, we site-selectively phosphorylated the activation loop of protein kinase p38α. Phosphorylation at natural (180) and unnatural (172) sites created two pure phospho-forms. p38α bearing only a single phosphocysteine (pCys) as a mimic of pThr at 180 was sufficient to switch the kinase to an active state, capable of processing natural protein substrate ATF2; 172 site phosphorylation did not. In this way, we chemically recapitulated triggering of a relevant segment of the MAPK-signaling pathway in vitro. This allowed detailed kinetic analysis of global and stoichiometric phosphorylation events catalyzed by p38α and revealed that site 180 is a sufficient activator alone and engenders dominant mono-phosphorylation activity. Moreover, a survey of kinase inhibition using inhibitors with different (Type I/II) modes (including therapeutically relevant) revealed unambiguously that Type II inhibitors inhibit phosphorylated p38α and allowed discovery of a predictive kinetic analysis based on cooperativity to distinguish Type I vs II.

  8. Multisite phosphorylation of spinach leaf sucrose-phosphate synthase

    SciTech Connect

    Huber, J.L.; Huber, S.C. )

    1990-05-01

    Spinach leaf sucrose-phosphate synthase is phosphorylated both in vivo and in vitro on serine residues. Phosphorylation of SPS in vivo yields twelve major phosphopeptides after a tryptic digest and two dimensional mapping. The in vivo labeling of three of these SPS P-peptides is reduced in illuminated leaves where the extracted enzyme is activated relative to that of dark leaves. Two of these inhibitory sites are phosphorylated as well when SPS is inactivated in vitro using ({sup 32}P)ATP. In vivo phosphorylation of two other sites is enhanced during mannose feeding of the leaves (in light or dark) which produces the highest activation state of SPS. Overall, the results confirm that light-dark regulation of SPS activity occurs as a result of regulatory seryl-phosphorylation and involves a balance between phosphorylation of sites which inhibit or stimulate activity. Regulation of the SPS protein kinase that inhibits activity is relatively unaffected by phosphate but inhibited by G1c 6-P (IC{sub 50}{approx}5 mM), which may explain the control of SPS activation state by light-dark signals.

  9. Processive phosphorylation of ERK MAP kinase in mammalian cells

    PubMed Central

    Aoki, Kazuhiro; Yamada, Masashi; Kunida, Katsuyuki; Yasuda, Shuhei; Matsuda, Michiyuki

    2011-01-01

    The mitogen-activated protein (MAP) kinase pathway is comprised of a three-tiered kinase cascade. The distributive kinetic mechanism of two-site MAP kinase phosphorylation inherently generates a nonlinear switch-like response. However, a linear graded response of MAP kinase has also been observed in mammalian cells, and its molecular mechanism remains unclear. To dissect these input-output behaviors, we quantitatively measured the kinetic parameters involved in the MEK (MAPK/ERK kinase)-ERK MAP kinase signaling module in HeLa cells. Using a numerical analysis based on experimentally determined parameters, we predicted in silico and validated in vivo that ERK is processively phosphorylated in HeLa cells. Finally, we identified molecular crowding as a critical factor that converts distributive phosphorylation into processive phosphorylation. We proposed the term quasi-processive phosphorylation to describe this mode of ERK phosphorylation that is operated under the physiological condition of molecular crowding. The generality of this phenomenon may provide a new paradigm for a diverse set of biochemical reactions including multiple posttranslational modifications. PMID:21768338

  10. Phosphorylation of Cytokinin by Adenosine Kinase from Wheat Germ 1

    PubMed Central

    Chen, Chong-Maw; Eckert, Richard L.

    1977-01-01

    Adenosine kinase was partially purified from wheat germ. This enzyme preparation, which was devoid of adenine phosphoribosyltransferase and nearly free of adenosine deaminase but contained adenylate kinase, rapidly phosphorylated adenosine and a cytokinin, N6-(δ2-isopentenyl)adenosine. Electrophoretic analysis indicated that only N6-(δ2-isopentenyl)adenosine-monophosphate was formed from the cytokinin while about 55% AMP, 45% ADP, and a trace of ATP were formed from adenosine. The biosynthesized nucleoside monophosphates were quantitatively hydrolyzed to the corresponding nucleosides by 5′-nucleotidase and the isopentenyl side chain of the phosphorylated cytokinin was not cleaved. The enzyme did not catalyze phosphorylation of inosine. The phosphorylation of the cytokinin and adenosine required ATP and Mg2+. The pH optimum was from 6.8 to 7.2 for both the cytokinin and adenosine. At pH 7 and 37 C the Km and Vmax for the cytokinin were 31 μm and 8.3 nmoles per mg protein per minute, and the values for adenosine were 8.7 μm and 46 nmoles per mg protein per minute. Crude enzyme preparations from tobacco callus tissue and wheat germ phosphorylated N6-(δ2-isopentenyl)adenosine. These preparations also phosphorylated N6-(δ2-isopentenyl)adenine when 5-phosphorylribose-1-pyrophosphate was present. PMID:16659870

  11. Cyclin B targets p34cdc2 for tyrosine phosphorylation.

    PubMed

    Meijer, L; Azzi, L; Wang, J Y

    1991-06-01

    A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.

  12. Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics.

    PubMed

    Bläsing, O E; Westhoff, P; Svensson, P

    2000-09-08

    C4 phosphoenolpyruvate carboxylases have evolved from ancestral C3 isoforms during the evolution of angiosperms and gained distinct kinetic and regulatory properties compared with the C3 isozymes. To identify amino acid residues and/or domains responsible for these C4-specific properties the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia (C4) was compared with its orthologue in the closely related C3 plant Flaveria pringlei. Reciprocal enzyme chimera were constructed and the kinetic constants, K(0.5) and k(cat), as well as the Hill coefficient, h, were determined for the substrate phosphoenolpyruvate both in the presence and absence of the activator glucose 6-phosphate. By this approach two regions were identified which determined most of the kinetic differences of the C4 and C3 ppcA phosphoenolpyruvate carboxylases with respect to the substrate PEP. In addition, the experiments suggest that the two regions do not act additively but interact with each other. The region between amino acids 296 and 437 is essential for activation by glucose 6-phosphate. The carboxyl-terminal segment between amino acids 645 and 966 contains a C4 conserved serine or a C3 invariant alanine at position 774 in the respective enzyme isoform. Site-directed mutagenesis shows that this position is a key determinant for the kinetic properties of the two isozymes.

  13. Squid neurofilaments. Phosphorylation and Ca2+-dependent proteolysis in situ.

    PubMed

    Brown, A; Eagles, P A

    1986-10-01

    Three major polypeptides co-purify with neurofilaments from squid (Loligo forbesi) axoplasm: P60 (apparent Mr 60,000), P200 (apparent Mr 200,000) and Band 1 (apparent Mr 400,000). Anti-IFA, a monoclonal antibody that recognizes an epitope common to all classes of intermediate filaments, binds to P200 and P60. When axoplasm is incubated with [32P]Pi, the major phosphorylated polypeptides are P200 and Band 1. We have investigated Ca2+-dependent proteolysis of [32P]phosphorylated axoplasm in order to localize the major sites of phosphorylation and to probe the arrangement of the polypeptides in the filament. The proteinase preferentially cleaves P200 and Band 1, liberating the phosphorylated domains. Analysis of proteolysed filaments by electron microscopy and gel electrophoresis shows that most of P200 and Band 1 can be cleaved while still maintaining intact filaments. We suggest that P200 is initially cleaved within a single highly sensitive region, generating two major fragments called P100p (apparent Mr 100,000) and P110s (apparent Mr 110,000). P100p contains the Anti-IFA epitope and co-sediments with filaments, whereas P110s is highly phosphorylated and does not sediment with filaments. Band 1 is cleaved to produce a soluble high-Mr fragment that is phosphorylated and that represents a major portion of the undigested component, whereas P60 is relatively resistant to limited proteolysis. Thus proteolysis appears to define two major filament domains: a conserved core that forms the backbone of the filament, and a highly phosphorylated peripheral region that is not essential for filament integrity.

  14. Phosphorylation of NLRC4 is critical for inflammasome activation.

    PubMed

    Qu, Yan; Misaghi, Shahram; Izrael-Tomasevic, Anita; Newton, Kim; Gilmour, Laurie L; Lamkanfi, Mohamed; Louie, Salina; Kayagaki, Nobuhiko; Liu, Jinfeng; Kömüves, László; Cupp, James E; Arnott, David; Monack, Denise; Dixit, Vishva M

    2012-10-25

    NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis. Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3×Flag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser 533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium). Western blotting with a NLRC4 phospho-Ser 533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4(-/-) macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKCδ) as a candidate NLRC4 kinase. Recombinant PKCδ phosphorylated NLRC4 S533 in vitro, immunodepletion of PKCδ from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro, and Prkcd(-/-) macrophages exhibited greatly attenuated caspase-1 activation and IL-1β secretion specifically in response to S. typhimurium. Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.

  15. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.

    PubMed

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2015-03-01

    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.

  16. Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity.

    PubMed

    Eaves, Deborah J; Haque, Tamanna; Tudor, Richard L; Barron, Yoshimi; Zampronio, Cleidiane G; Cotton, Nicholas P J; de Graaf, Barend H J; White, Scott A; Cooper, Helen J; Franklin, F Christopher H; Harper, Jeffery F; Franklin-Tong, Vernonica E

    2017-03-01

    Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important posttranslational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollen-expressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plant Papaver rhoeas were inhibited by phosphorylation. Despite the potential significance, there is a paucity of data on sPPase phosphorylation and regulation. Here, we used liquid chromatographic tandem mass spectrometry to map phosphorylation sites to the otherwise divergent amino-terminal extensions on these pollen sPPases. Despite the absence of reports in the literature on mapping phosphorylation sites on sPPases, a database survey of various proteomes identified a number of examples, suggesting that phosphorylation may be a more widely used mechanism to regulate these enzymes. Phosphomimetic mutants of Pr-p26.1a/b significantly and differentially reduced PPase activities by up to 2.5-fold at pH 6.8 and 52% in the presence of Ca(2+) and hydrogen peroxide over unmodified proteins. This indicates that phosphoregulation of key sites can inhibit the catalytic responsiveness of these proteins in concert with key intracellular events. As sPPases are essential for many metabolic pathways in eukaryotic cells, our findings identify the phosphorylation of sPPases as a potential master regulatory mechanism that could be used to attenuate metabolism.

  17. Regulation of divalent metal transporter-1 by serine phosphorylation

    PubMed Central

    Seo, Young Ah; Kumara, Ruvin; Wetli, Herbert; Wessling-Resnick, Marianne

    2016-01-01

    Divalent metal transporter-1 (DMT1) mediates dietary iron uptake across the intestinal mucosa and facilitates peripheral delivery of iron released by transferrin in the endosome. Here, we report that classical cannabinoids (Δ9-tetrahydrocannabinol, Δ9-THC), nonclassical cannabinoids (CP 55,940), aminoalkylindoles (WIN 55,212-2) and endocannabinoids (anandamide) reduce 55Fe and 54Mn uptake by HEK293T(DMT1) cells stably expressing the transporter. siRNA knockdown of cannabinoid receptor type 2 (CB2) abrogated inhibition. CB2 is a G-protein (GTP-binding protein)-coupled receptor that negatively regulates signal transduction cascades involving serine/threonine kinases. Immunoprecipitation experiments showed that DMT1 is serine-phosphorylated under basal conditions, but that treatment with Δ9-THC reduced phosphorylation. Site-directed mutation of predicted DMT1 phosphosites further showed that substitution of serine with alanine at N-terminal position 43 (S43A) abolished basal phosphorylation. Concordantly, both the rate and extent of 55Fe uptake in cells expressing DMT1(S43A) was reduced compared with those expressing wild-type DMT1. Among kinase inhibitors that affected DMT1-mediated iron uptake, staurosporine also reduced DMT1 phosphorylation confirming a role for serine phosphorylation in iron transport regulation. These combined data indicate that phosphorylation at serine 43 of DMT1 promotes transport activity, whereas dephosphorylation is associated with loss of iron uptake. Since anti-inflammatory actions mediated through CB2 would be associated with reduced DMT1 phosphorylation, we postulate that this pathway provides a means to reduce oxidative stress by limiting iron uptake. PMID:27681840

  18. Abundant protein phosphorylation potentially regulates Arabidopsis anther development

    PubMed Central

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-01-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana. However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4–7 and 8–12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  19. A signature of the oxygenase intermediate in catalysis by ribulose-bisphosphate carboxylase/oxygenase as provided by a site-directed mutant.

    PubMed

    Chen, Y R; Hartman, F C

    1995-05-19

    An uncharacterized minor transient product, observed in our earlier studies of substrate turnover by the E48Q mutant of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase (Lee, E. H., Harpel, M. R., Chen, Y.-R., and Hartman, F. C. (1993) J. Biol. Chem. 268, 26583-26591), becomes a major product when it is trapped and stabilized with borate as an additive to the reaction mixture. Chemical characterization establishes this novel product as D-glycero-2,3-pentodiulose 1,5-bisphosphate, thereby demonstrating oxidation of the C-3 hydroxyl of D-ribulose 1,5-bisphosphate to a carbonyl. As the formation of the novel oxidation product is oxygen-dependent and generates hydrogen peroxide, its precursor must be a peroxy derivative of ribulose bisphosphate. Thus, discovery of the dicarbonyl bisphosphate lends direct support to the long standing, but heretofore unproven, postulate that the normal pathway for oxidative cleavage of ribulose bisphosphate by the wild-type enzyme entails a peroxy intermediate. Our results also suggest that stabilization of the peroxy intermediate by the wild-type enzyme promotes carbon-carbon scission as opposed to elimination of hydrogen peroxide.

  20. Assembly of in vitro synthesized large subunits into ribulose-bisphosphate carboxylase/oxygenase. Formation and discharge of an L8-like species.

    PubMed

    Hubbs, A E; Roy, H

    1993-06-25

    Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) from higher plants consists of eight approximately 53-kDa large subunits and eight approximately 14-kDa small subunits. Cytosolic ribosomes synthesize the small subunits as precursors, which enter the chloroplast, undergo proteolytic processing, and assemble with large subunits. Large subunits, synthesized in the chloroplast, first form a complex with the chloroplast chaperonin 60 (Cpn60(14)). In the presence of ATP, large subunits dissociate from Cpn60(14) and assemble into Rubisco. We now describe partial characterization of a new species, Z, containing radiotracer-labeled, newly synthesized pea Rubisco large subunits. Rubisco assembly occurs in low salt in the presence of small subunits and ATP. As with Rubisco assembly, the formation of Z is ATP-dependent and is inhibited by high chloride. Once formed, Z is stable except in high chloride. Z does not appear to interact directly with small subunits. However, after Z formation, Rubisco assembly occurs in an ATP-independent reaction that requires KCl and small subunits. These results are consistent with the hypothesis that Z is a large subunit containing structure that can contribute large subunits to Rubisco under appropriate conditions. Z shares some physical characteristics with reported cyanobacterial L8 core particles. However, formation of Rubisco from Z in the absence of ATP and the presence of small subunits appears to require conditions that otherwise destabilize Z.

  1. Structural characterization and the determination of negative cooperativity in the tight binding of 2-carboxyarabinitol bisphosphate to higher plant ribulose bisphosphate carboxylase.

    PubMed

    Johal, S; Partridge, B E; Chollet, R

    1985-08-15

    When CO2/Mg2+-activated spinach leaf ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) is incubated with the transition-state analog 2-carboxyarabinitol 1,5-bisphosphate, an essentially irreversible complex is formed. The extreme stability of this quaternary complex has allowed the use of native analytical isoelectric focusing, anion-exchange chromatography, and nondenaturing polyacrylamide gel electrophoresis to probe the mechanism of the binding process and the effects of ligand tight-binding on the structure of the protein molecule. Changes in the chromatographic and electrophoretic properties of the enzyme upon tight binding of the inhibitor reveal that the ligand induces a conformational reorganization which extends to the surface of the protein molecule and, at saturation, results in a 16% decrease in apparent molecular weight. Analysis of ligand binding by isoelectric focusing shows that (i) incubating the protein with a stoichiometric molar concentration of ligand (site basis) results in an apparently charge homogeneous enzyme population with an isoelectric point of 4.9, and (ii) substoichiometric levels of ligand produce differential effects on each of the charge microheterogeneous native enzyme forms. These stoichiometry-dependent changes in electrofocusing band patterns were employed as a probe of cooperativity in the ligand tight-binding process. The tight-binding reaction was shown to be negatively cooperative.

  2. Susceptibility of podocytes to palmitic acid is regulated by fatty acid oxidation and inversely depends on acetyl-CoA carboxylases 1 and 2.

    PubMed

    Kampe, Kapil; Sieber, Jonas; Orellana, Jana Marina; Mundel, Peter; Jehle, Andreas Werner

    2014-02-15

    Type 2 diabetes is characterized by dyslipidemia with elevated free fatty acids (FFAs). Loss of podocytes is a hallmark of diabetic nephropathy, and podocytes are susceptible to saturated FFAs, which induce endoplasmic reticulum (ER) stress and podocyte death. Genome-wide association studies indicate that expression of acetyl-CoA carboxylase (ACC) 2, a key enzyme of fatty acid oxidation (FAO), is associated with proteinuria in type 2 diabetes. Here, we show that stimulation of FAO by aminoimidazole-4-carboxamide-1β-D-ribofuranoside (AICAR) or by adiponectin, activators of the low-energy sensor AMP-activated protein kinase (AMPK), protects from palmitic acid-induced podocyte death. Conversely, inhibition of carnitine palmitoyltransferase (CPT-1), the rate-limiting enzyme of FAO and downstream target of AMPK, augments palmitic acid toxicity and impedes the protective AICAR effect. Etomoxir blocked the AICAR-induced FAO measured with tritium-labeled palmitic acid. The beneficial effect of AICAR was associated with a reduction of ER stress, and it was markedly reduced in ACC-1/-2 double-silenced podocytes. In conclusion, the stimulation of FAO by modulating the AMPK-ACC-CPT-1 pathway may be part of a protective mechanism against saturated FFAs that drive podocyte death. Further studies are needed to investigate the potentially novel therapeutic implications of these findings.

  3. Electrophoretic assay for ribulose 1,5-bisphosphate carboxylase/oxygenase in guard cells and other leaf cells of Vicia faba L

    SciTech Connect

    Tarczynski, M.C.; Outlaw, W.H. Jr.; Arold, N.; Neuhoff, V.; Hampp, R. Max-Planck-Institute fuer Experimentelle Medizin, Goettingen Universitaet Tuebingen )

    1989-04-01

    The ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents of guard cells and other cells of Vicia faba L. leaflet were determined. To prevent proteolysis, proteins of frozen protoplast preparations or of cells excised from freeze-dried leaf were extracted directly in a sodium-dodecyl-sulfate-containing solution which was heated immediately after sample addition. Protein profiles of the different cell types were obtained by electrophoresis of the extracts and subsequent densitometry of the stained protein bands. About one-third of the protein of palisade parenchyma and of spongy parenchyma was Rubisco large subunit. Using chlorophyll (Chl):protein ratios previously obtained, we calculate mesophyll contained ca. 22 millimoles Rubisco per mole Chl. In contrast, guard-cell protoplast preparations were calculated to contain from 0.7 to 2.2 millimoles Rubisco per mole Chl. The upper end of this range is an overestimate resulting from contamination by mesophyll and to the method of peak integration. Extracts of excised guard cells were calculated to contain 0.05 to 0.17 millimole Rubisco per mole Chl. We conclude that Rubisco is absent, or virtually so, in guard cells of V. faba.

  4. Decreasing the Rate of Metabolic Ketone Reduction in the Discovery of a Clinical Acetyl-CoA Carboxylase Inhibitor for the Treatment of Diabetes

    PubMed Central

    2015-01-01

    Acetyl-CoA carboxylase (ACC) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus (T2DM), hepatic steatosis, and cancer. However, the identification of tool compounds suitable to test the hypothesis in human trials has been challenging. An advanced series of spirocyclic ketone-containing ACC inhibitors recently reported by Pfizer were metabolized in vivo by ketone reduction, which complicated human pharmacology projections. We disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl. Incorporation of weakly basic functionality improved solubility and led to the identification of 9 as a clinical candidate for the treatment of T2DM. Phase I clinical studies demonstrated dose-proportional increases in exposure, single-dose inhibition of de novo lipogenesis (DNL), and changes in indirect calorimetry consistent with increased whole-body fatty acid oxidation. This demonstration of target engagement validates the use of compound 9 to evaluate the role of DNL in human disease. PMID:25423286

  5. Effect of CO{sub 2} concentration on carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase expression in pea

    SciTech Connect

    Majeau, N.; Coleman, J.R.

    1996-10-01

    The effect of external CO{sub 2} concentration on the expression of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was examined in pea (Pisum sativum cv Little Marvel) leaves. Enzyme activities and their transcript levels were reduced in plants grown at 1000 {mu}L/L CO{sub 2} compared with plants grown in ambient air. Growth at 160 {mu}L/L CO{sub 2} also appeared to reduce steady-state transcript levels for the rbcS, the gene encoding the small subunit of Rubisco, and for ca, the gene encoding CA; however, rbcS transcripts were reduced to a greater extent at this concentration. Rubisco activity was slightly lower in plants grown at 160 {mu}L/L CO{sub 2}, and CA activity was significantly higher than that observed in air-grown plants. Transfer of plants from 1000 {mu}L/L to air levels of CO{sub 2} resulted in a rapid increase in both ca and rbcS transcript abundance in fully expanded leaves, followed by an increase in enzyme activity. Plants transferred from air to high-CO{sub 2} concentrations appeared to modulate transcript abundance and enzyme activity less quickly. Foliar carbohydrate levels were also examined in plants grown continuously at high and ambient CO{sub 2}, and following changes in growth conditions that rapidly altered ca and rbcS transcript abundance and enzyme activities. 39 refs., 2 figs., 3 tabs.

  6. Diel Rhythms in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Glutamine Synthetase Gene Expression in a Natural Population of Marine Picoplanktonic Cyanobacteria (Synechococcus spp.)

    PubMed Central

    Wyman, Michael

    1999-01-01

    Diel periodicity in the expression of key genes involved in carbon and nitrogen assimilation in marine Synechococcus spp. was investigated in a natural population growing in the surface waters of a cyclonic eddy in the northeast Atlantic Ocean. Synechococcus sp. cell concentrations within the upper mixed layer showed a net increase of three- to fourfold during the course of the experiment (13 to 22 July 1991), the population undergoing approximately one synchronous division per day. Consistent with the observed temporal pattern of phycoerythrin (CpeBA) biosynthesis, comparatively little variation was found in cpeBA mRNA abundance during either of the diel cycles investigated. In marked contrast, the relative abundance of transcripts originating from the genes encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL) and glutamine synthetase (glnA) showed considerable systematic temporal variation and oscillated during the course of each diel cycle in a reciprocal rhythm. Whereas activation of rbcL transcription was clearly not light dependent, expression of glnA appeared sensitive to endogenous changes in the physiological demands for nitrogen that arise as a natural consequence of temporal periodicity in photosynthetic carbon assimilation. The data presented support the hypothesis that a degree of temporal separation may exist between the most active periods of carbon and nitrogen assimilation in natural populations of marine Synecoccoccus spp. PMID:10427062

  7. Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria.

    PubMed

    Whitney, Spencer M; Sharwood, Robert E; Orr, Douglas; White, Sarah J; Alonso, Hernan; Galmés, Jeroni

    2011-08-30

    Improving global yields of important agricultural crops is a complex challenge. Enhancing yield and resource use by engineering improvements to photosynthetic carbon assimilation is one potential solution. During the last 40 million years C(4) photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO(2)-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C(3) ancestors, C(4) plants combine a faster rubisco with a biochemical CO(2)-concentrating mechanism, enabling more efficient use of water and nitrogen and enhanced yield. Here we show the versatility of plastome manipulation in tobacco for identifying sequences in C(4)-rubisco that can be transplanted into C(3)-rubisco to improve carboxylation rate (V(C)). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C(3)), Flaveria floridana (C(3)-C(4)), and Flaveria bidentis (C(4)), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C(4) ((309)Ile; faster V(C), lower CO(2) affinity) and C(3) ((309)Met; slower V(C), higher CO(2) affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco V(C) in C(3) crops. Coengineering a catalytically faster C(3) rubisco and a CO(2)-concentrating mechanism within C(3) crop species could enhance their efficiency in resource use and yield.

  8. Preliminary X-ray crystallographic study of wild-type and mutant ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii.

    PubMed

    Yen, A; Haas, E J; Selbo, K M; Ross 2nd, C R; Spreitzer, R J; Stezowski, J J

    1998-07-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase is the key enzyme for photosynthesis. The wild-type and mutant (amino-acid substitutions in the catalytically important loop 6 region) enzymes from Chlamydomonas reinhardtii, a unicellular green alga, were crystallized. Wild-type, single-mutant (V331A) and two double-mutant (V331A/T342I and V331A/G344S) proteins were activated with cofactors CO2 and Mg2+, complexed with the substrate analog 2'-carboxyarabinitol-1,5-bisphosphate, and crystallized in apparently isomorphous forms. Unit-cell determinations have been completed for three of the enzymes. They display orthorhombic symmetry with similar cell parameters: wild type a = 130.4, b = 203. 3, c = 208.5 A; single mutant (V331A) a = 128.0, b = 203.0, c = 207. 0A; and double mutant (V331A/T342I) a = 130.0, b = 202.1, c = 209.7 A. Crystals of the wild-type and single-mutant (V331A) enzymes diffracted to approximately 2.8 A. A small crystal of the double-mutant (V331A/T342I) enzyme diffracted to approximately 6 A. A partial data set (68% complete) of the wild-type protein has been collected at room temperature to about 3.5 A.

  9. Diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes in the MgCl2-dominated deep hypersaline anoxic basin discovery.

    PubMed

    van der Wielen, Paul W J J

    2006-06-01

    Partial sequences of the form I (cbbL) and form II (cbbM) of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit genes were obtained from the brine and interface of the MgCl2-dominated deep hypersaline anoxic basin Discovery. CbbL and cbbM genes were found in both brine and interface of the Discovery Basin but were absent in the overlying seawater. The diversity of both genes in the brine and interface was low, which might caused by the extreme saline conditions in Discovery of approximately 5 M MgCl2. None of the retrieved sequences were closely related to sequences deposited in the GenBank database. A phylogenetic analysis demonstrated that the cbbL sequences were affiliated with a Thiobacillus sp. or with one of the RuBisCO genes from Hydrogenovibrio marinus. The cbbM sequences clustered with thiobacilli or formed a new group with no close relatives. The results implicate that bacteria with the potential for carbon dioxide fixation and chemoautotrophy are present in the Discovery Basin. This is the first report demonstrating that RuBisCO genes are present under hypersaline conditions of 5 M MgCl2.

  10. Quantitative analyses of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes (cbbL) in typical paddy soils.

    PubMed

    Xiao, Ke-Qing; Bao, Peng; Bao, Qiong-Li; Jia, Yan; Huang, Fu-Yi; Su, Jian-Qiang; Zhu, Yong-Guan

    2014-01-01

    The Calvin cycle is known to be the major pathway for CO2 fixation, but our current understanding of its occurrence and importance in paddy soils is poor. In this study, the diversity of three ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbLG, cbbLR, cbbM) was investigated by clone library, T-RFLP, qPCR, and enzyme assay in five paddy soils in China. The cbbLG sequences revealed a relatively low level of diversity and were mostly related to the sequences of species from Thiobacillus. In contrast, highly diverse cbbLR and cbbM sequences were dispersed on the phylogenetic trees, and most of them were distantly related to known sequences, even forming separate clusters. Abundances of three cbbL genes ranged from 10(6) to 10(9) copies g(-1) soil, and cbbLR outnumbered cbbM and cbbLG in all soil samples, indicating that cbbLR may play a more important role than other two cbbL genes. Soil properties significantly influenced cbbL diversity in five paddy soils, of which clay content, C/N ratio, CEC, pH, and SOC correlated well with variations in microbial composition and abundance. In summary, this study provided a comparison of three cbbL genes, advancing our understanding of their role in carbon sequestration and nutrient turnover in the paddy soil.

  11. RNAi knockdown of acetyl-CoA carboxylase gene eliminates jinggangmycin-enhanced reproduction and population growth in the brown planthopper, Nilaparvata lugens

    PubMed Central

    Zhang, Yi-Xin; Ge, Lin-Quan; Jiang, Yi-Ping; Lu, Xiu-Li; Li, Xin; Stanley, David; Song, Qi-Sheng; Wu, Jin-Cai

    2015-01-01

    A major challenge in ecology lies in understanding the coexistence of intraguild species, well documented at the organismal level, but not at the molecular level. This study focused on the effects of the antibiotic, jinggangmycin (JGM), a fungicide widely used in Asian rice agroecosystems, on reproduction of insects within the planthopper guild, including the brown planthopper (BPH) Nilaparvata lugens and the white-backed planthopper (WBPH) Sogatella furcifera, both serious resurgence rice pests. JGM exposure significantly increased BPH fecundity and population growth, but suppressed both parameters in laboratory and field WBPH populations. We used digital gene expression and transcriptomic analyses to identify a panel of differentially expressed genes, including a set of up-regulated genes in JGM-treated BPH, which were down-regulated in JGM-treated WBPH. RNAi silencing of Acetyl Co-A carboxylase (ACC), highly expressed in JGM-treated BPH, reduced ACC expression (by > 60%) and eliminated JGM-induced fecundity increases in BPH. These findings support our hypothesis that differences in ACC expression separates intraguild species at the molecular level. PMID:26482193

  12. Regulation of Ribulose-1,5-Bisphosphate Carboxylase Expression in Second Leaves of Maize Seedlings from Low and High Yield Populations 1

    PubMed Central

    Loza-Tavera, Herminia; Martínez-Barajas, Eleazar; Sánchez-de-Jiménez, Estela

    1990-01-01

    Ribulose-1,5-bisphosphate carboxylase oxygenase (EC 4.1.1.39) (Rubisco) activity, Rubisco-protein, and Rubisco large and small subunit gene (rbcL and rbcS) transcripts were measured at seven stages of development in the second leaf of maize (Zea mays L.) seedlings belonging to low and high yield populations. During the three early stages of development, when the leaf has not yet expanded, it was determined that increments in Rubisco-activity were caused by increases in Rubisco-protein and its mRNAs. Afterward, the rbcS level decreased sharply down to nondetectable levels at the seventh stage, when the leaf was at the beginning of senescence. As a contrast, rbcL transcript decreased slowly and Rubisco-protein accumulated up to the fifth stage, when the leaf reached its maximum expansion. A slight decrease in Rubisco-protein was then observed. These results suggest that at early stages of development Rubisco-activity and Rubisco-protein are regulated mainly at the transcriptional level. At the later phase the regulation seems to be at other biochemical levels. Neither Rubisco activity nor Rubisco-protein showed correlation with yield for both maize populations at this stage of development. Slightly higher levels of both transcripts were observed in the high yield population. Images Figure 1 Figure 6 PMID:16667500

  13. The MDM2–p53–pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic β-cells

    PubMed Central

    Li, Xiaomu; Cheng, Kenneth K. Y.; Liu, Zhuohao; Yang, Jin-Kui; Wang, Baile; Jiang, Xue; Zhou, Yawen; Hallenborg, Philip; Hoo, Ruby L. C.; Lam, Karen S. L.; Ikeda, Yasuhiro; Gao, Xin; Xu, Aimin

    2016-01-01

    Mitochondrial metabolism is pivotal for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. However, little is known about the molecular machinery that controls the homeostasis of intermediary metabolites in mitochondria. Here we show that the activation of p53 in β-cells, by genetic deletion or pharmacological inhibition of its negative regulator MDM2, impairs GSIS, leading to glucose intolerance in mice. Mechanistically, p53 activation represses the expression of the mitochondrial enzyme pyruvate carboxylase (PC), resulting in diminished production of the TCA cycle intermediates oxaloacetate and NADPH, and impaired oxygen consumption. The defective GSIS and mitochondrial metabolism in MDM2-null islets can be rescued by restoring PC expression. Under diabetogenic conditions, MDM2 and p53 are upregulated, whereas PC is reduced in mouse β-cells. Pharmacological inhibition of p53 alleviates defective GSIS in diabetic islets by restoring PC expression. Thus, the MDM2–p53–PC signalling axis links mitochondrial metabolism to insulin secretion and glucose homeostasis, and could represent a therapeutic target in diabetes. PMID:27265727

  14. Photosynthetic CO2 fixation and ribulose bisphosphate carboxylase/oxygenase activity of Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus.

    PubMed Central

    Steinberg, N A; Meeks, J C

    1989-01-01

    The cyanobacterium Nostoc sp. strain UCD 7801, immediately after separation from pure cultures of a reconstituted symbiotic association with the bryophyte Anthoceros punctatus, exhibited a rate of light-dependent CO2 fixation that was eightfold lower than that measured in the free-living growth state. Ribulose bisphosphate carboxylase/oxygenase (RuBPC/O) specific activity was also eightfold lower in cell extracts of symbiotic strain 7801 relative to that in free-living cultures. The in vitro activity from symbiotic strain 7801 could not be increased by incubation under the standard RuBPC/O activation conditions. Polyclonal antibodies against the RuBPC/O large subunit were used in an enzyme-linked immunosorbent assay to determine that RuBPC/O accounted for 4.3 and 5.2% of the total protein in cell extracts of strain 7801 grown in symbiotic and free-living states, respectively. The results imply that the regulation of RuBPC/O activity in the symbiotic growth state is by a posttranslational mechanism rather than by an alteration in RuBPC/O protein synthesis. The amount of carboxyarabinitol bisphosphate required to irreversibly inhibit RuBPC/O activity of sybiotic cell extracts was 80% of that required for extracts of free-living cultures. This result indicates that any covalent modification of RuBPC/O in symbiotically associated Nostoc cells did not interfere with the ribulose bisphosphate binding site, since inactive enzyme also bound carboxyarabinitol bisphosphate. Images PMID:2509431

  15. Mapping of a locus correcting lack of phosphoribosylaminoimidazole carboxylase activity in Chinese hamster ovary cell Ade-D mutants to human chromosome 4.

    PubMed

    Barton, J W; Hart, I M; Patterson, D

    1991-02-01

    The human phosphoribosylaminoimidazole (AIR) carboxylase locus has been until this report one of the genes encoding purine biosynthetic enzymes that had not been assigned to an individual human chromosome. Characterization of Chinese hamster ovary (CHO) cell mutant Ade-D showed that the cell line was unable to produce IMP and accumulated AIR. CHO Ade-D cells were fused with normal human lymphocytes utilizing inactivated Sendai virus and the resulting hybrid cell lines were selected for purine prototrophy. Cytogenetic analysis showed a 100% concordance value for chromosome 4. Two of the isolated subclones contained only the long arm of chromosome 4 translocated onto a CHO chromosome, providing evidence for a regional assignment of the Ade-D gene to the long arm of chromosome 4. Two of the subclones containing chromosome 4 were subjected to the BrdU visible light segregation. All of the isolated purine auxotrophic cell lines showed a loss of the q arm of chromosome 4. The localization of the Ade-D locus to the long arm of chromosome 4 may reveal further clustering of the mammalian purine genes since the Ade-A locus has previously been regionally assigned to 4pter-q21.

  16. Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small cell lung cancer in preclinical models

    PubMed Central

    Svensson, Robert U.; Parker, Seth J.; Eichner, Lillian J.; Kolar, Matthew J.; Wallace, Martina; Brun, Sonja N.; Lombardo, Portia S.; Van Nostrand, Jeanine L.; Hutchins, Amanda; Vera, Lilliana; Gerken, Laurie; Greenwood, Jeremy; Bhat, Sathesh; Harriman, Geraldine; Westlin, William F.; Harwood, H. James; Saghatelian, Alan; Kapeller, Rosana; Metallo, Christian M.; Shaw, Reuben J.

    2016-01-01

    Continuous de novo fatty acid synthesis is a common feature of cancer required to meet the biosynthetic demands of a growing tumor. This process is controlled by the rate-limiting enzyme acetyl-CoA carboxylase (ACC), an attractive but traditionally intractable drug target. Here, we provide genetic and pharmacological evidence that in preclinical models ACC is required to maintain de novo fatty acid synthesis needed for growth and viability of non-small cell lung cancer (NSCLC). We describe the ability of ND-646—an allosteric inhibitor of the ACC enzymes ACC1 and ACC2 that prevents ACC subunit dimerization—to suppress fatty acid synthesis in vitro and in vivo. Chronic ND-646 treatment of xenograft and genetically engineered mouse models of NSCLC inhibited tumor growth. When administered as a single agent or in combination with the standard-of-care drug carboplatin, ND-646 markedly suppressed lung tumor growth in the Kras;Trp53−/− (also known as KRAS p53) and Kras;Stk11−/− (also known as KRAS Lkb1) mouse models of NSCLC. These findings demonstrate that ACC mediates a metabolic liability of NSCLC and that ACC inhibition by ND-646 is detrimental to NSCLC growth, supporting further examination of the use of ACC inhibitors in oncology. PMID:27643638

  17. Comparison of Patterns of Accumulation of Ribulose Bisphosphate Carboxylase Antigen and Catalytic Activity and Measurement of Antigen Half-Life during the Cell Cycle of Chlorella sorokiniana1

    PubMed Central

    Toman, P. David; Schmidt, Robert R.

    1985-01-01

    By use of specific immunochemical procedures, ribulose-1,5-bisphosphate carboxylase (RuBPCase), antigen and catalytic activity were shown to have coincident step-patterns of accumulation during the cell cycle of Chlorella sorokiniana. Pulse-chase studies, employing radioactive sulfate, were performed during the period of rapid accumulation of enzyme activity and during the period of constant enzyme activity in the cell cycle. No degradation of RuBPCase antigen could be detected during either of these cell cycle periods. Thus, the step-pattern of accumulation of RuBPCase activity resulted from periodic synthesis of an enzyme that was stable under steady-state cell cycle conditions. Although inhibition of protein synthesis by cycloheximide, at different times in the cell cycle in the light, resulted in rapid decay of RuBPCase activity, this loss in activity occurred without detectable loss in enzyme antigen. When synchronous cells were placed into the dark, to slow the rate of protein synthesis in the absence of cycloheximide, the levels of enzyme antigen and activity decreased by 30 and 50%, respectively, during the 10-hour dark period. Thus, in C. sorokiniana changes in RuBPCase activity do not necessarily reflect parallel changes in enzyme antigen, particularly when cell growth is perturbed by changes from steady-state cultural conditions. PMID:16664496

  18. The glossyhead1 Allele of ACC1 Reveals a Principal Role for Multidomain Acetyl-Coenzyme A Carboxylase in the Biosynthesis of Cuticular Waxes by Arabidopsis

    SciTech Connect

    Lu, S.; Xu, C.; Zhao, H.; Parsons, E. P.; Kosma, D. K.; Xu, X.; Chao, D.; Lohrey, G.; Bangarusamy, D. K.; Wang, G.; Bressan, R. A.; Jenks, M. A.

    2011-11-01

    A novel mutant of Arabidopsis (Arabidopsis thaliana), having highly glossy inflorescence stems, postgenital fusion in floral organs, and reduced fertility, was isolated from an ethyl methanesulfonate-mutagenized population and designated glossyhead1 (gsd1). The gsd1 locus was mapped to chromosome 1, and the causal gene was identified as a new allele of Acetyl-Coenzyme A Carboxylase1 (ACC1), a gene encoding the main enzyme in cytosolic malonyl-coenzyme A synthesis. This, to our knowledge, is the first mutant allele of ACC1 that does not cause lethality at the seed or early germination stage, allowing for the first time a detailed analysis of ACC1 function in mature tissues. Broad lipid profiling of mature gsd1 organs revealed a primary role for ACC1 in the biosynthesis of the very-long-chain fatty acids (C{sub 20:0} or longer) associated with cuticular waxes and triacylglycerols. Unexpectedly, transcriptome analysis revealed that gsd1 has limited impact on any lipid metabolic networks but instead has a large effect on environmental stress-responsive pathways, especially senescence and ethylene synthesis determinants, indicating a possible role for the cytosolic malonyl-coenzyme A-derived lipids in stress response signaling.

  19. RNAi knockdown of acetyl-CoA carboxylase gene eliminates jinggangmycin-enhanced reproduction and population growth in the brown planthopper, Nilaparvata lugens.

    PubMed

    Zhang, Yi-Xin; Ge, Lin-Quan; Jiang, Yi-Ping; Lu, Xiu-Li; Li, Xin; Stanley, David; Song, Qi-Sheng; Wu, Jin-Cai

    2015-10-20

    A major challenge in ecology lies in understanding the coexistence of intraguild species, well documented at the organismal level, but not at the molecular level. This study focused on the effects of the antibiotic, jinggangmycin (JGM), a fungicide widely used in Asian rice agroecosystems, on reproduction of insects within the planthopper guild, including the brown planthopper (BPH) Nilaparvata lugens and the white-backed planthopper (WBPH) Sogatella furcifera, both serious resurgence rice pests. JGM exposure significantly increased BPH fecundity and population growth, but suppressed both parameters in laboratory and field WBPH populations. We used digital gene expression and transcriptomic analyses to identify a panel of differentially expressed genes, including a set of up-regulated genes in JGM-treated BPH, which were down-regulated in JGM-treated WBPH. RNAi silencing of Acetyl Co-A carboxylase (ACC), highly expressed in JGM-treated BPH, reduced ACC expression (by > 60%) and eliminated JGM-induced fecundity increases in BPH. These findings support our hypothesis that differences in ACC expression separates intraguild species at the molecular level.

  20. Nuclear-Cytoplasmic Conflict in Pea (Pisum sativum L.) Is Associated with Nuclear and Plastidic Candidate Genes Encoding Acetyl-CoA Carboxylase Subunits

    PubMed Central

    Bogdanova, Vera S.; Zaytseva, Olga O.; Mglinets, Anatoliy V.; Shatskaya, Natalia V.; Kosterin, Oleg E.; Vasiliev, Gennadiy V.

    2015-01-01

    In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized. PMID:25789472

  1. Expression of recombinant cytoplasmic yeast pyruvate carboxylase for the improvement of the production of human erythropoietin by recombinant BHK-21 cells.

    PubMed

    Irani, Noushin; Beccaria, Alejandro José; Wagner, Roland

    2002-02-28

    Recently, a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to reconstitute the missing link between glycolysis and TCA, thus increasing the flux of glucose into the TCA and resulting in a higher intracellular ATP content. Now, these metabolically engineered cells have been additionally transfected with a plasmid bearing the gene for human erythropoietin. EPO yield and substrate-specific productivity of the recombinant BHK-21 cells have been compared to control cells without the PYC2-gene but transfected with the plasmid coding for the expression of the selection genes and EPO. PYC2-expressing clones showed a 2-fold higher glucose-specific productivity and a 2-fold higher product concentration in a continuously perfused bioreactor. Moreover, the PYC2 expression enabled the cells to become more resistant to low glucose concentrations in the culture medium. They could produce at nearly maximum productivity under glucose-limiting conditions of 0.05-1 gl(-1) that guaranteed a reduced accumulation of lactate in fed-batch production systems. Due to the fact that PYC2-expressing cells are characterized by reduced glucose consumption, a prolonged production phase in bioreactors can be maintained. Based on the demand not to fall short of 80% cell viability for the production, EPO could be produced for 2 days (30%) longer compared to the control due to a more economic exploitation of glucose, and the prolonged viability period of the cells using a batch cultivation driven by glutamine limitation.

  2. Decreasing the Rate of Metabolic Ketone Reduction in the Discovery of a Clinical Acetyl-CoA Carboxylase Inhibitor for the Treatment of Diabetes

    SciTech Connect

    Griffith, David A.; Kung, Daniel W.; Esler, William P.; Amor, Paul A.; Bagley, Scott W.; Beysen, Carine; Carvajal-Gonzalez, Santos; Doran, Shawn D.; Limberakis, Chris; Mathiowetz, Alan M.; McPherson, Kirk; Price, David A.; Ravussin, Eric; Sonnenberg, Gabriele E.; Southers, James A.; Sweet, Laurel J.; Turner, Scott M.; Vajdos, Felix F.

    2014-12-26

    We found that Acetyl-CoA carboxylase (ACC) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus (T2DM), hepatic steatosis, and cancer. However, the identification of tool compounds suitable to test the hypothesis in human trials has been challenging. An advanced series of spirocyclic ketone-containing ACC inhibitors recently reported by Pfizer were metabolized in vivo by ketone reduction, which complicated human pharmacology projections. Here, we disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl. Incorporation of weakly basic functionality improved solubility and led to the identification of 9 as a clinical candidate for the treatment of T2DM. Phase I clinical studies demonstrated dose-proportional increases in exposure, single-dose inhibition of de novo lipogenesis (DNL), and changes in indirect calorimetry consistent with increased whole-body fatty acid oxidation. This demonstration of target engagement validates the use of compound 9 to evaluate the role of DNL in human disease.

  3. CO2 reduction and organic compounds production by photosynthetic bacteria with surface displayed carbonic anhydrase and inducible expression of phosphoenolpyruvate carboxylase.

    PubMed

    Park, Ju-Yong; Kim, Yang-Hoon; Min, Jiho

    2017-01-01

    In Rhodobacter sphaeroides, carbonic anhydrase (CA; EC 4.2.1.1) is a zinc-containing metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-) while phosphoenolpyruvate carboxylase (PEPC; 4.1.1.31), an enzyme involved in the carbon metabolism that catalyzed the fixation of CO2 to PEP, is a key factor for biological fixation of CO2 and enhances the production of organic compounds. In this study, the recombinant R. sphaeroides with highly-expressed CA was developed based on a surface displayed system of CA (pJY-OmpCA) on the outer membrane of R. sphaeroides using outer membrane protein (Omp) in R. sphaeroides, Finally, two more different recombinant R. sphaeroides were developed, which transformed with a two-vector system harboring cytosolic expressed CA (pJY-OmpCA-CA)or PEPC (pJY-OMPCA-PEPC) in R. sphaeroides with surface displayed CA on the outer membrane. In case of recombinant R. sphaeroides with the pJY-OmpCA-PEPC, it has shown the highest CO2 reduction efficiency and the production of several organic compounds (carotenoids, polyhydroxybutyrate, malic acid, succinic acid). It means that the surface displayed CA on the R. sphaeroides would accelerate the CO2-bicabonate conversion on the bacterial outer membrane. Moreover, inducible over-expression of PEPC with surface-displayed CA was successfully used to facilitate a rapider CO2 reduction and quicker production of organic compounds.

  4. A potential role for RNA turnover in the light regulation of plant gene expression: ribulose-1,5-bisphosphate carboxylase small subunit in soybean.

    PubMed Central

    Shirley, B W; Meagher, R B

    1990-01-01

    Post-transcriptional regulation of the genes encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase was examined in soybean seedlings. Substantial discrepancies were detected between relative in vitro transcription rates and steady-state RNA levels in light- and dark-grown seedling leaves, indicating that rbcS RNA may be degraded more rapidly in light than in darkness. Additional data imply that the turnover mechanism is rapidly induced by light, maintained for some time in darkness, and that it may be negatively controlled by far-red light. The proposed RNA turnover system does not affect all RNAs equally since a soybean actin gene showed equivalent in vitro transcription rates and RNA levels in light and darkness. Soybean rbcS genes may be subject to a novel mode of control in which light-induced expression is accompanied by an increased rate of RNA degradation. Models for the specific regulation of rbcS RNA stability in response to light are presented. Images PMID:2356127

  5. Engineering the α-ketoglutarate overproduction from raw glycerol by overexpression of the genes encoding NADP+-dependent isocitrate dehydrogenase and pyruvate carboxylase in Yarrowia lipolytica.

    PubMed

    Yovkova, Venelina; Otto, Christina; Aurich, Andreas; Mauersberger, Stephan; Barth, Gerold

    2014-03-01

    To establish and develop a biotechnological process of α-ketoglutaric acid (KGA) production by Yarrowia lipolytica, it is necessary to increase the KGA productivity and to reduce the amounts of by-products, e.g. pyruvic acid (PA) as major by-product and fumarate, malate and succinate as minor by-products. The aim of this study was the improvement of KGA overproduction with Y. lipolytica by a gene dose-dependent overexpression of genes encoding NADP(+)-dependent isocitrate dehydrogenase (IDP1) and pyruvate carboxylase (PYC1) under KGA production conditions from the renewable carbon source raw glycerol. Recombinant Y. lipolytica strains were constructed, which harbour multiple copies of the respective IDP1, PYC1 or IDP1 and PYC1 genes together. We demonstrated that a selective increase in IDP activity in IDP1 multicopy transformants changes the produced amount of KGA. Overexpression of the gene IDP1 in combination with PYC1 had the strongest effect on increasing the amount of secreted KGA. About 19% more KGA compared to strain H355 was produced in bioreactor experiments with raw glycerol as carbon source. The applied cultivation conditions with this strain significantly reduced the main by-product PA and increased the KGA selectivity to more than 95% producing up to 186 g l(-1) KGA. This proved the high potential of this multicopy transformant for developing a biotechnological KGA production process.

  6. Inhibition of Acetyl-CoA Carboxylase 1 (ACC1) and 2 (ACC2) Reduces Proliferation and De Novo Lipogenesis of EGFRvIII Human Glioblastoma Cells

    PubMed Central

    Jones, Jessica E. C.; Esler, William P.; Patel, Rushi; Lanba, Adhiraj; Vera, Nicholas B.; Pfefferkorn, Jeffrey A.; Vernochet, Cecile

    2017-01-01

    Tumor cell proliferation and migration processes are regulated by multiple metabolic pathways including glycolysis and de novo lipogenesis. Since acetyl-CoA carboxylase (ACC) is at the junction of lipids synthesis and oxidative metabolic pathways, we investigated whether use of a dual ACC inhibitor would provide a potential therapy against certain lipogenic cancers. The impact of dual ACC1/ACC2 inhibition was investigated using a dual ACC1/ACC2 inhibitor as well as dual siRNA knock down on the cellular viability and metabolism of two glioblastoma multiform cancer cell lines, U87 and a more aggressive form, U87 EGFRvIII. We first demonstrated that while ACCi inhibited DNL in both cell lines, ACCi preferentially blunted the U87 EGFRvIII cellular proliferation capacity. Metabolically, chronic treatment with ACCi significantly upregulated U87 EGFRvIII cellular respiration and extracellular acidification rate, a marker of glycolytic activity, but impaired mitochondrial health by reducing maximal respiration and decreasing mitochondrial ATP production efficiency. Moreover, ACCi treatment altered the cellular lipids content and increased apoptotic caspase activity in U87 EGFRvIII cells. Collectively these data indicate that ACC inhibition, by reducing DNL and increasing cellular metabolic rate, may have therapeutic utility for the suppression of lipogenic tumor growth and warrants further investigation. PMID:28081256

  7. Increased phosphorylation of the NR1 subunit of the NMDA receptor following cerebral ischemia.

    PubMed

    Cheung, H H; Teves, L; Wallace, M C; Gurd, J W

    2001-09-01

    The effects of transient cerebral ischemia on phosphorylation of the NR1 subunit of the NMDA receptor by protein kinase C (PKC) and protein kinase A (PKA) were investigated. Adult rats received 15 min of cerebral ischemia followed by various times of recovery. Phosphorylation was examined by immunoblotting hippocampal homogenates with antibodies that recognized NR1 phosphorylated on the PKC phosphorylation sites Ser890 and Ser896, the PKA phosphorylation site Ser897, or dually phosphorylated on Ser896 and Ser897. The phosphorylation of all sites examined increased following ischemia. The increase in phosphorylation by PKC was greater than by PKA. The ischemia-induced increase in phosphorylation was predominantly associated with the population of NR1 that was insoluble in 1% deoxycholate. Enhanced phosphorylation of NR1 by PKC and PKA may contribute to alterations in NMDA receptor function in the postischemic brain.

  8. Akt phosphorylates and regulates Pdcd4 tumor suppressor protein.

    PubMed

    Palamarchuk, Alexey; Efanov, Alexey; Maximov, Vadim; Aqeilan, Rami I; Croce, Carlo M; Pekarsky, Yuri

    2005-12-15

    Programmed cell death 4 (Pdcd4) is a tumor suppressor protein that interacts with eukaryotic initiation factor 4A and inhibits protein synthesis. Pdcd4 also suppresses the transactivation of activator protein-1 (AP-1)-responsive promoters by c-Jun. The Akt (protein kinase B) serine/threonine kinase is a key mediator of phosphoinositide 3-kinase pathway involved in the regulation of cell proliferation, survival, and growth. Because Pdcd4 has two putative Akt phosphorylation sites at Ser(67) and Ser(457), we investigated whether Akt phosphorylates and regulates Pdcd4. Our results show that Akt specifically phosphorylates Ser(67) and Ser(457) residues of Pdcd4 in vitro and in vivo. We further show that phosphorylation of Pdcd4 by Akt causes nuclear translocation of Pdcd4. Using luciferase assay, we show that phosphorylation of Pdcd4 by Akt also causes a significant decrease of the ability of Pdcd4 to interfere with the transactivation of AP-1-responsive promoter by c-Jun.

  9. Akt phosphorylates Tal1 oncoprotein and inhibits its repressor activity.

    PubMed

    Palamarchuk, Alexey; Efanov, Alexey; Maximov, Vadim; Aqeilan, Rami I; Croce, Carlo M; Pekarsky, Yuri

    2005-06-01

    The helix-loop-helix transcription factor Tal1 is required for blood cell development and its activation is a frequent event in T-cell acute lymphoblastic leukemia. The Akt (protein kinase B) kinase is a key player in transduction of antiapoptotic and proliferative signals in T cells. Because Tal1 has a putative Akt phosphorylation site at Thr90, we investigated whether Akt regulates Tal1. Our results show that Akt specifically phosphorylates Thr90 of the Tal1 protein within its transactivation domain in vitro and in vivo. Coimmunoprecipitation experiments showed the presence of Tal1 in Akt immune complexes, suggesting that Tal1 and Akt physically interact. We further showed that phosphorylation of Tal1 by Akt causes redistribution of Tal1 within the nucleus. Using luciferase assay, we showed that phosphorylation of Tal1 by Akt decreased repressor activity of Tal1 on EpB42 (P4.2) promoter. Thus, these data indicate that Akt interacts with Tal1 and regulates Tal1 by phosphorylation at Thr90 in a phosphatidylinositol 3-kinase-dependent manner.

  10. Histamine-stimulated phosphorylation of gastric parietal cell proteins

    SciTech Connect

    Chew, C.S.; Brown, M.R.

    1987-05-01

    Parietal cells from rabbit gastric mucosa respond to histamine with increased HCl secretion. Histamine also increases cAMP and activates cAMP-dependent protein kinase(s) in these cells. cAMP analogues and forskolin appear to mimic these effects. More recently histamine and forskolin but not cAMP-stimulated increases in (Ca/sup 2 +/)/sub i/ have been detected in parietal cells enriched to 98 +/- 2% (n=10) purity using a combined Nycodenz density gradient/centrifugal elutriation technique. In the present experiments parietal cells were loaded with /sup 32/P to label ATP pools then stimulated with histamine or chlorophenylthio-cAMP plus the H/sub 2/ receptor antagonist, cimetidine. Total cell extracts were separated via 2D-gel electrophoresis and analyzed with a Masscomp computer and PDQuest software. Results indicate that histamine stimulates phosphorylation of at least two proteins with molecular weights 49 and 33 kDa and respective pI's of 6.4 and 6.0. Changes in phosphorylation are detected within 1 min of stimulation and remain elevated for at least 15 min. No change in specific activity of samples was detected during this time. A third protein also showed increased phosphorylation but the response appeared more transient. They conclude that histamine increases phosphorylation of several parietal cell proteins via a cAMP-dependent mechanism. The relationship between changes in phosphorylation and onset of HCl secretion remains to be determined.

  11. Tau phosphorylation affects its axonal transport and degradation

    PubMed Central

    Rodríguez-Martín, Teresa; Cuchillo-Ibáñez, Inmaculada; Noble, Wendy; Nyenya, Fanon; Anderton, Brian H.; Hanger, Diane P.

    2013-01-01

    Phosphorylated forms of microtubule-associated protein tau accumulate in neurofibrillary tangles in Alzheimer's disease. To investigate the effects of specific phosphorylated tau residues on its function, wild type or phosphomutant tau was expressed in cells. Elevated tau phosphorylation decreased its microtubule binding and bundling, and increased the number of motile tau particles, without affecting axonal transport kinetics. In contrast, reducing tau phosphorylation enhanced the amount of tau bound to microtubules and inhibited axonal transport of tau. To determine whether differential tau clearance is responsible for the increase in phosphomimic tau, we inhibited autophagy in neurons which resulted in a 3-fold accumulation of phosphomimic tau compared with wild type tau, and endogenous tau was unaffected. In autophagy-deficient mouse embryonic fibroblasts, but not in neurons, proteasomal degradation of phosphomutant tau was also reduced compared with wild type tau. Therefore, autophagic and proteasomal pathways are involved in tau degradation, with autophagy appearing to be the primary route for clearing phosphorylated tau in neurons. Defective autophagy might contribute to the accumulaton of tau in neurodegenerative diseases. PMID:23601672

  12. Structural changes accompanying phosphorylation of tarantula muscle myosin filaments

    PubMed Central

    1987-01-01

    Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg- ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin. PMID:2958483

  13. Queuine mediated inhibition in phosphorylation of tyrosine phosphoproteins in cancer.

    PubMed

    Pathak, Chandramani; Jaiswal, Yogesh K; Vinayak, Manjula

    2008-09-01

    Protein phosphorylation or dephosphorylation is the most important regulatory switch of signal transduction contributing to control of cell proliferation. The reversibility of phosphorylation and dephosphorylation is due to the activities of kinases and phosphatase, which determine protein phosphorylation level of cell under different physiological and pathological conditions. Receptor tyrosine kinase (RTK) mediated cellular signaling is precisely coordinated and tightly controlled in normal cells which ensures regulated mitosis. Deregulation of RTK signaling resulting in aberrant activation in RTKs leads to malignant transformation. Queuine is one of the modified base of tRNA which participates in down regulation of tyrosine kinase activity. The guanine analogue queuine is a nutrient factor to eukaryotes and occurs as free base or modified nucleoside queuosine into the first anticodon position of specific tRNAs. The tRNAs are often queuine deficient in cancer and fast proliferating tissues. The present study is aimed to investigate queuine mediated inhibition in phosphorylation of tyrosine phosphorylated proteins in lymphoma bearing mouse. The result shows high level of cytosolic and membrane associated tyrosine phosphoprotein in DLAT cancerous mouse liver compared to normal. Queuine treatments down regulate the level of tyrosine phosphoproteins, which suggests that queuine is involved in regulation of mitotic signaling pathways.

  14. Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy.

    PubMed

    Qian, Xu; Li, Xinjian; Cai, Qingsong; Zhang, Chuanbao; Yu, Qiujing; Jiang, Yuhui; Lee, Jong-Ho; Hawke, David; Wang, Yugang; Xia, Yan; Zheng, Yanhua; Jiang, Bing-Hua; Liu, David X; Jiang, Tao; Lu, Zhimin

    2017-03-02

    Autophagy is crucial for maintaining cell homeostasis. However, the precise mechanism underlying autophagy initiation remains to be defined. Here, we demonstrate that glutamine deprivation and hypoxia result in inhibition of mTOR-mediated acetyl-transferase ARD1 S228 phosphorylation, leading to ARD1-dependent phosphoglycerate kinase 1 (PGK1) K388 acetylation and subsequent PGK1-mediated Beclin1 S30 phosphorylation. This phosphorylation enhances ATG14L-associated class III phosphatidylinositol 3-kinase VPS34 activity by increasing the binding of phosphatidylinositol to VPS34. ARD1-dependent PGK1 acetylation and PGK1-mediated Beclin1 S30 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumorigenesis. Furthermore, PGK1 K388 acetylation levels correlate with Beclin1 S30 phosphorylation levels and poor prognosis in glioblastoma patients. Our study unearths an important mechanism underlying cellular-stress-induced autophagy initiation in which the protein kinase activity of the metabolic enzyme PGK1 plays an instrumental role and reveals the significance of the mutual regulation of autophagy and cell metabolism in maintaining cell homeostasis.

  15. Paxillin-kinase-linker tyrosine phosphorylation regulates directional cell migration.

    PubMed

    Yu, Jianxin A; Deakin, Nicholas O; Turner, Christopher E

    2009-11-01

    Directed cell migration requires the coordination of growth factor and cell adhesion signaling and is of fundamental importance during embryonic development, wound repair, and pathological conditions such as tumor metastasis. Herein, we demonstrate that the ArfGAP, paxillin-kinase-linker (PKL/GIT2), is tyrosine phosphorylated in response to platelet-derived growth factor (PDGF) stimulation, in an adhesion dependent manner and is necessary for directed cell migration. Using a combination of pharmacological inhibitors, knockout cells and kinase mutants, FAK, and Src family kinases were shown to mediate PDGF-dependent PKL tyrosine phosphorylation. In fibroblasts, expression of a PKL mutant lacking the principal tyrosine phosphorylation sites resulted in loss of wound-induced cell polarization as well as directional migration. PKL phosphorylation was necessary for PDGF-stimulated PKL binding to the focal adhesion protein paxillin and expression of paxillin or PKL mutants defective in their respective binding motifs recapitulated the polarization defects. RNA interference or expression of phosphorylation mutants of PKL resulted in disregulation of PDGF-stimulated Rac1 and PAK activities, reduction of Cdc42 and Erk signaling, as well as mislocalization of betaPIX. Together these studies position PKL as an integral component of growth factor and cell adhesion cross-talk signaling, controlling the development of front-rear cell polarity and directional cell migration.

  16. Reciprocal Phosphorylation and Palmitoylation Control Dopamine Transporter Kinetics*

    PubMed Central

    Moritz, Amy E.; Rastedt, Danielle E.; Stanislowski, Daniel J.; Shetty, Madhur; Smith, Margaret A.; Vaughan, Roxanne A.; Foster, James D.

    2015-01-01

    The dopamine transporter is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the brain. Dopamine transporter function is regulated by protein kinase C (PKC) and other signaling pathways through mechanisms that are complex and poorly understood. Here we investigate the role of Ser-7 phosphorylation and Cys-580 palmitoylation in mediating steady-state transport kinetics and PKC-stimulated transport down-regulation. Using both mutational and pharmacological approaches, we demonstrate that these post-translational modifications are reciprocally regulated, leading to transporter populations that display high phosphorylation-low palmitoylation or low phosphorylation-high palmitoylation. The balance between the modifications dictates transport capacity, as conditions that promote high phosphorylation or low palmitoylation reduce transport Vmax and enhance PKC-stimulated down-regulation, whereas conditions that promote low phosphorylation or high palmitoylation increase transport Vmax and suppress PKC-stimulated down-regulation. Transitions between these functional states occur when endocytosis is blocked or undetectable, indicating that the modifications kinetically regulate the velocity of surface transporters. These findings reveal a novel mechanism for control of DA reuptake that may represent a point of dysregulation in DA imbalance disorders. PMID:26424792

  17. Inhibition of peptide aggregation by means of enzymatic phosphorylation

    PubMed Central

    Folmert, Kristin; Broncel, Malgorzata; v. Berlepsch, Hans; Ullrich, Christopher Hans; Siegert, Mary-Ann

    2016-01-01

    As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP-dependent protein kinase) that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β-sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated. PMID:28144314

  18. 2-(4-Bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, a new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. Determination of reaction parameters and characterization of an active site peptide.

    PubMed

    Herndon, C S; Hartman, F C

    1984-03-10

    A new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, has been prepared, Reductive amination of ribulose-P2 with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography on quaternary aminoethyl-Sephadex. Subsequent bromoacetylation of the isolated amino bisphosphates gave reagents A and B (ribo and arabino epimers of 2-(4-bromoacetamido) anilino-2-deoxypentitol 1,5-bisphosphate) which were competitive inhibitors of the carboxylase with Ki values of 705 and 104 microM, respectively. Reagent A exhibited no time-dependent effects on the carboxylase in either the deactivated or activated state. Incubation of the enzyme with reagent B in the presence of the essential activators CO2 and Mg2+, however, resulted in an irreversible, time-dependent loss of activity, with a Kinact of 125 microM and a minimal half-time of 7.3 min. Covalent incorporation of [14C]reagent B was directly proportional to the loss of activity, with total inactivation correlating with an incorporation of 1.1 mol of reagent/mol of subunit. Inclusion of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate protected against inactivation with a concomitant reduction in incorporation. Neither reagent affected the activity of spinach carboxylase. Fractionation of [14C]reagent B-modified enzyme on DEAE-cellulose, subsequent to carboxymethylation and tryptic digestion, revealed two major radioactive peaks of approximately equal area. Digestion of each peak with alkaline phosphatase and rechromatography on DEAE-cellulose resulted in pure peptides I and II. The peptides were identical except in the site of labeling: peptide I contained a modified cysteinyl residue while peptide II contained a modified histidyl residue. Automated Edman degradation established the sequence as (sequence in text) which is located near the NH2 terminus

  19. Biodistribution, kinetics, and efficacy of highly phosphorylated and non-phosphorylated beta-glucuronidase in the murine model of mucopolysaccharidosis VII.

    PubMed

    Sands, M S; Vogler, C A; Ohlemiller, K K; Roberts, M S; Grubb, J H; Levy, B; Sly, W S

    2001-11-16

    Enzyme replacement therapy (ERT) has been shown to be effective at reducing the accumulation of undegraded substrates in lysosomal storage diseases. Most ERT studies have been performed with recombinant proteins that are mixtures of phosphorylated and non-phosphorylated enzyme. Because different cell types use different receptors to take up phosphorylated or non-phosphorylated enzyme, it is difficult to determine which form of enzyme contributed to the clinical response. Here we compare the uptake, distribution, and efficacy of highly phosphorylated and non-phosphorylated beta-glucuronidase (GUSB) in the MPS VII mouse. Highly phosphorylated murine GUSB was efficiently taken up by a wide range of tissues. In contrast, non-phosphorylated murine GUSB was taken up primarily by tissues of the reticuloendothelial (RE) system. Although the tissue distribution was different, the half-lives of both enzymes in any particular tissue were similar. Both preparations of enzyme were capable of preventing the accumulation of lysosomal storage in cell types they targeted. An important difference in clinical efficacy emerged in that phosphorylated GUSB was more efficient than non-phosphorylated enzyme at preventing the hearing loss associated with this disease. These data suggest that both forms of enzyme contribute to the clinical responses of ERT in MPS VII mice but that enzyme preparations containing phosphorylated GUSB are more broadly effective than non-phosphorylated enzyme.

  20. Regulatory Phosphorylation of Ikaros by Bruton's Tyrosine Kinase

    PubMed Central

    Zhang, Jian; Ishkhanian, Rita; Uckun, Fatih M.

    2013-01-01

    Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK) as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4) within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros. PMID:23977012

  1. Ultrasensitive dual phosphorylation dephosphorylation cycle kinetics exhibits canonical competition behavior

    NASA Astrophysics Data System (ADS)

    Huang, Qingdao; Qian, Hong

    2009-09-01

    We establish a mathematical model for a cellular biochemical signaling module in terms of a planar differential equation system. The signaling process is carried out by two phosphorylation-dephosphorylation reaction steps that share common kinase and phosphatase with saturated enzyme kinetics. The pair of equations is particularly simple in the present mathematical formulation, but they are singular. A complete mathematical analysis is developed based on an elementary perturbation theory. The dynamics exhibits the canonical competition behavior in addition to bistability. Although widely understood in ecological context, we are not aware of a full range of biochemical competition in a simple signaling network. The competition dynamics has broad implications to cellular processes such as cell differentiation and cancer immunoediting. The concepts of homogeneous and heterogeneous multisite phosphorylation are introduced and their corresponding dynamics are compared: there is no bistability in a heterogeneous dual phosphorylation system. A stochastic interpretation is also provided that further gives intuitive understanding of the bistable behavior inside the cells.

  2. Protein kinase C coordinates histone H3 phosphorylation and acetylation

    PubMed Central

    Darieva, Zoulfia; Webber, Aaron; Warwood, Stacey; Sharrocks, Andrew D

    2015-01-01

    The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly. DOI: http://dx.doi.org/10.7554/eLife.09886.001 PMID:26468616

  3. Foxp3 drives oxidative phosphorylation and protection from lipotoxicity

    PubMed Central

    Cobbold, Stephen Paul; Adams, Elizabeth; Necula, Andra Stefania; Zhang, Wei; Huang, Honglei; Thomas, Benjamin; Hester, Svenja S.; Betz, Alexander G.

    2017-01-01

    Tregs can adopt a catabolic metabolic program with increased capacity for fatty acid oxidation–fueled oxidative phosphorylation (OXPHOS). It is unclear why this form of metabolism is favored in Tregs and, more specifically, whether this program represents an adaptation to the environment and developmental cues or is “hardwired” by Foxp3. Here we show, using metabolic analysis and an unbiased mass spectroscopy–based proteomics approach, that Foxp3 is both necessary and sufficient to program Treg-increased respiratory capacity and Tregs’ increased ability to utilize fatty acids to fuel oxidative phosphorylation. Foxp3 drives upregulation of components of all the electron transport complexes, increasing their activity and ATP generation by oxidative phosphorylation. Increased fatty acid β-oxidation also results in selective protection of Foxp3+ cells from fatty acid–induced cell death. This observation may provide novel targets for modulating Treg function or selection therapeutically. PMID:28194435

  4. Modification of EWS/WT1 functional properties by phosphorylation

    PubMed Central

    Kim, Jungho; Lee, Joseph M.; Branton, Philip E.; Pelletier, Jerry

    1999-01-01

    In many human cancers, tumor-specific chromosomal rearrangements are known to create chimeric products with the ability to transform cells. The EWS/WT1 protein is such a fusion product, resulting from a t(11;22) chromosomal translocation in desmoplastic small round cell tumors, where 265 aa from the EWS amino terminus are fused to the DNA binding domain of the WT1 tumor suppressor gene. Herein, we find that EWS/WT1 is phosphorylated in vivo on serine and tyrosine residues and that this affects DNA binding and homodimerization. We also show that EWS/WT1 can interact with, and is a substrate for, modification on tyrosine residues by c-Abl. Tyrosine phosphorylation of EWS/WT1 by c-Abl negatively regulates its DNA binding properties. These results indicate that the biological activity of EWS/WT1 is closely linked to its phosphorylation status. PMID:10588700

  5. Crystal Structure of a Phosphorylation-coupled Saccharide Transporter

    SciTech Connect

    Y Cao; X Jin; E Levin; H Huang; Y Zong; W Hendrickson; J Javitch; K Rajashankar; M Zhou; et al.

    2011-12-31

    Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

  6. Phosphorylation of lamins determine their structural properties and signaling functions

    PubMed Central

    Torvaldson, Elin; Kochin, Vitaly; Eriksson, John E

    2015-01-01

    Lamin A/C is part of the nuclear lamina, a meshwork of intermediate filaments underlying the inner nuclear membrane. The lamin network is anchoring a complex set of structural and linker proteins and is either directly or through partner proteins also associated or interacting with a number of signaling protein and transcription factors. During mitosis the nuclear lamina is dissociated by well established phosphorylation- dependent mechanisms. A-type lamins are, however, also phosphorylated during interphase. A recent study identified 20 interphase phosphorylation sites on lamin A/C and explored their functions related to lamin dynamics; movements, localization and solubility. Here we discuss these findings in the light of lamin functions in health and disease. PMID:25793944

  7. Partial high-resolution structure of phosphorylated and non-phosphorylated leucine-rich amelogenin protein adsorbed to hydroxyapatite

    SciTech Connect

    Masica, David L.; Gray, Jeffrey J.; Shaw, Wendy J.

    2011-07-21

    The formation of biogenic materials requires the interaction of organic molecules with the mineral phase. In forming enamel, the amelogenin proteins contribute to the mineralization of hydroxyapatite (HAp). Leucine-rich amelogenin protein (LRAP) is a naturally occurring splice variant of amelogenin that comprises amelogenin’s predicted HAp binding domains. We determined the partial structure of phosphorylated and non-phosphorylated LRAP variants bound to HAp using combined solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. The ssNMR measurements indicate a largely extended structure for both variants, though some measurements are consistent with a partially helical N-terminal segment. Structure prediction was biased using 21 ssNMR measurements at five HAp crystal faces. The predicted fold of LRAP is similar at all HAp faces studied, regardless of phosphorylation. LRAP’s predicted structure is relatively extended with a helix-turn-helix motif in the N-terminal domain and some helix in the C-terminal domain. The N-terminal domain of the phosphorylated variant binds HAp more tightly than the N-terminal domain of the non-phosphorylated variant. Both variants are predicted to preferentially bind the {010} HAp crystal face providing further evidence that amelogenins block crystal growth on the a and b faces to allow elongated crystals in the c-axis. Pacific Northwest National Laboratory is operated by Battelle for the US Department of Energy.

  8. P2X7 receptors stimulate AKT phosphorylation in astrocytes

    PubMed Central

    Jacques-Silva, Maria C; Rodnight, Richard; Lenz, Guido; Liao, Zhongji; Kong, Qiongman; Tran, Minh; Kang, Yuan; Gonzalez, Fernando A; Weisman, Gary A; Neary, Joseph T

    2004-01-01

    Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X7 subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. P2Y and P2X receptor agonists ATP, uridine 5′-triphosphate (UTP) and 2′,3′-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X7 receptor. Activation was maximal at 5 – 10 min and was sustained for 60 min; the EC50 for BzATP was approximately 50 μM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X7 receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5′-phosphate-6-azophenyl-2′,4′-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. In conclusion, our data indicate that stimulation of astrocytic P2X7 receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism. PMID:15023862

  9. Mechanism of APC/CCDC20 activation by mitotic phosphorylation

    PubMed Central

    Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G.; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A.; Brunner, Michael R.; Davidson, Iain F.; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A.; Peters, Jan-Michael

    2016-01-01

    Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis. PMID:27114510

  10. Regulation of Rho proteins by phosphorylation in the cardiovascular system.

    PubMed

    Loirand, Gervaise; Guilluy, Christophe; Pacaud, Pierre

    2006-08-01

    The small G protein Rho signaling pathways are recognized as major regulators of cardiovascular functions, and activation of Rho proteins appears to be a common component for the pathogenesis of hypertension and vascular proliferative disorders. Rho proteins are tightly regulated, and recent evidence suggests that modulation of Rho protein signaling by phosphorylation of Rho proteins provides an additional simple mechanism for coordinating Rho protein functions. This regulation by phosphorylation is particularly important in the arterial wall, where RhoA protein expressed in vascular smooth muscle cells is controlled by the endothelium through the nitric oxide/cGMP-dependent kinase pathway.

  11. Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

    PubMed Central

    Sharma, Mayuri; Bender, Brian J.; Kamil, Jeremy P.; Lye, Ming F.; Pesola, Jean M.; Reim, Natalia I.; Hogle, James M.

    2014-01-01

    ABSTRACT Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC. IMPORTANCE Human cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the

  12. Phosphorylation and recruitment of Syk by immunoreceptor tyrosine-based activation motif-based phosphorylation of tamalin.

    PubMed

    Hirose, Masayuki; Kitano, Jun; Nakajima, Yoshiaki; Moriyoshi, Koki; Yanagi, Shigeru; Yamamura, Hirohei; Muto, Takanori; Jingami, Hisato; Nakanishi, Shigetada

    2004-07-30

    Tamalin is a scaffold protein that forms a multiple protein assembly including metabotropic glutamate receptors (mGluRs) and several postsynaptic and protein-trafficking scaffold proteins in distinct mode of protein-protein association. In the present investigation, we report that tamalin possesses a typical immunoreceptor tyrosine-based activation motif (ITAM), which enables Syk kinase to be recruited and phosphorylated by the Src family kinases. Coimmunoprecipitation analysis of rat brain membrane fractions showed that tamalin is present in a multimolecular protein assembly comprising not only mGluR1 but also c-Src, Fyn, and a protein phosphatase, SHP-2. The protein association of both tamalin and c-Src, as determined by truncation analysis of mGluR1 in COS-7 cells, occurred at the carboxyl-terminal tail of mGluR1. Mutation analysis of tyrosine with phenylalanine in COS-7 cells revealed that paired tyrosines at the ITAM sequence of tamalin are phosphorylated preferentially by c-Src and Fyn, and this phosphorylation can recruit Syk kinase and enables it to be phosphorylated by the Src family kinases. The phosphorylated tyrosines at the ITAM sequence of tamalin were highly susceptible to dephosphorylation by protein-tyrosine phosphatases in COS-7 cells. Importantly, tamalin was endogenously phosphorylated and associated with Syk in retinoic acid-treated P19 embryonal carcinoma cells that undergo neuron-like differentiation. The present investigation demonstrates that tamalin is a novel signaling molecule that possesses a PDZ domain and a PDZ binding motif and mediates Syk signaling in an ITAM-based fashion.

  13. Ozone-induced ethylene emission accelerates the loss of ribulose-1,5-bisphosphate carboxylase/oxygenase and nuclear-encoded mRNAs in senescing potato leaves

    SciTech Connect

    Glick, R.E.; Schlagnhaufer, C.D.; Arteca, R.N.

    1995-11-01

    The relationships among O{sub 3}-induced accelerated senescence, induction of ethylene, and changes in specific mRNA and protein levels were investigated in potato (Solanum tuberosum L. cv Norland) plants. When plants were exposed to 0.08 {mu}L L{sup -1} O{sub 3} for 5 h d{sup -1}, steady-state levels of rbcS mRNA declined at least 5-fold in expanding leaves after 3 d of O{sub 3} exposure and ethylene levels increased 6- to 10-fold. The expression of OIP-1, a 1-aminocyclo-propane-1-carboxylate synthase cDNA from potato, correlated with increased production of ethylene and decreased levels of rbcS mRNA in foliage of plants treated with O{sub 3}. In plants exposed to 0.30 {mu}L L{sup -1} O{sub 3} for 4 h, rbcS transcript levels were reduced 4-fold, whereas nuclear run-on experiments revealed that rbcS mRNA may be due, in part, to posttranscriptional regulation. The levels of transcripts for other chloroplast proteins, glyceraldehyde-3-phosphate dehydrogenase, and a photosystem II chlorophyll a/b-binding protein decreased in O{sub 3}-treated plants, in parallel with the decrease in rbcS mRNA. The steady-state mRNA level of a cytosolic glyceraldehyde-3-phosphate dehydrogenase increased in O{sub 3}-treated plants. The induction of ethylene and changes in transcript levels preceded visible leaf damage and decreases in ribulose-1,5-biphosphate carboxylase/oxygenase protein levels. 40 refs., 6 figs.

  14. Determination of methylmalonyl coenzyme A by ultra high-performance liquid chromatography tandem mass spectrometry for measuring propionyl coenzyme A carboxylase activity in patients with propionic acidemia.

    PubMed

    Gotoh, Kana; Nakajima, Yoko; Tajima, Go; Watanabe, Yoriko; Hotta, Yuji; Kataoka, Tomoya; Kawade, Yoshihiro; Sugiyama, Naruji; Ito, Tetsuya; Kimura, Kazunori; Maeda, Yasuhiro

    2017-03-01

    Propionic acidemia (PA) is an inherited metabolic disease caused by low activity of propionyl coenzyme A (CoA) carboxylase (PCC), which metabolizes propionyl-CoA into methylmalonyl-CoA. Although many patients with PA have been identified by tandem mass spectrometry since the test was first included in neonatal mass screening in the 1990s, the disease severity varies. Thus, determining the specific level of PCC activity is considered to be helpful to grasp the severity of PA. We developed a new PCC assay method by the determination of methylmalonyl-CoA, which is formed by an enzyme reaction using peripheral lymphocytes, based on ultra high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). With methylmalonyl-CoA concentrations of 0.05, 0.5, and 5μmol/L, the intra-assay coefficients of variation (CVs) were 8.2%, 8.7%, and 5.1%, respectively, and the inter-assay CVs were 13.6%, 10.5%, and 5.9%, respectively. The PCC activities of 20 healthy individuals and 6 PA patients were investigated with this assay. Methylmalonyl-CoA was not detected in one PA patient with a severe form of the disease, but the remaining PA patients with mild disease showed residual activities (3.3-7.8%). These results demonstrate that determination of PCC activity with this assay would be useful to distinguish between mild and severe cases of PA to help choose an appropriate treatment plan.

  15. Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate kinase of the Triticum/Aegilops complex and the evolutionary history of polyploid wheat

    PubMed Central

    Huang, Shaoxing; Sirikhachornkit, Anchalee; Su, Xiujuan; Faris, Justin; Gill, Bikram; Haselkorn, Robert; Gornicki, Piotr

    2002-01-01

    The classic wheat evolutionary history is one of adaptive radiation of the diploid Triticum/Aegilops species (A, S, D), genome convergence and divergence of the tetraploid (Triticum turgidum AABB, and Triticum timopheevii AAGG) and hexaploid (Triticum aestivum, AABBDD) species. We analyzed Acc-1 (plastid acetyl-CoA carboxylase) and Pgk-1 (plastid 3-phosphoglycerate kinase) genes to determine phylogenetic relationships among Triticum and Aegilops species of the wheat lineage and to establish the timeline of wheat evolution based on gene sequence comparisons. Triticum urartu was confirmed as the A genome donor of tetraploid and hexaploid wheat. The A genome of polyploid wheat diverged from T. urartu less than half a million years ago (MYA), indicating a relatively recent origin of polyploid wheat. The D genome sequences of T. aestivum and Aegilops tauschii are identical, confirming that T. aestivum arose from hybridization of T. turgidum and Ae. tauschii only 8,000 years ago. The diploid Triticum and Aegilops progenitors of the A, B, D, G, and S genomes all radiated 2.5–4.5 MYA. Our data suggest that the Acc-1 and Pgk-1 loci have different histories in different lineages, indicating genome mosaicity and significant intraspecific differentiation. Some loci of the S genome of Aegilops speltoides and the G genome of T. timophevii are closely related, suggesting the same origin of some parts of their genomes. None of the Aegilops genomes analyzed is a close relative of the B genome, so the diploid progenitor of the B genome remains unknown. PMID:12060759

  16. Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales, Phaeophyta)

    NASA Astrophysics Data System (ADS)

    Shao, Zhanru; Liu, Fuli; Li, Qiuying; Yao, Jianting; Duan, Delin

    2014-03-01

    Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments, and consequently may have highly efficient ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity for carbon assimilation. In our study, we cloned the full-length Rubisco gene from S. japonica ( SJ-rbc). It contained an open reading frame for a large subunit gene ( SJ — rbcL) of 1 467 bp, a small subunit gene ( SJ-rbcS) of 420 bp, and a SJ-rbcL/S intergenic spacer of 269 bp. The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa, 5.81 and 15.84 kDa, 4.71, respectively. After induction with 1 mmol/L isopropyl- β-D-thiogalactopyranoside for 5 h and purification by Ni2+ affinity chromatography, electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein. Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night. This observation implied that Rubisco played a role in normal gametophytic growth and development. In juvenile sporophytes, mRNA levels of SJ-rbcL, carbonic anhydrase, Calvin-Benson-Bassham cycle-related enzyme, and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance. Similarly, expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m2·s). Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.

  17. ATP and magnesium promote cotton short-form ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase hexamer formation at low micromolar concentrations.

    PubMed

    Kuriata, Agnieszka M; Chakraborty, Manas; Henderson, J Nathan; Hazra, Suratna; Serban, Andrew J; Pham, Tuong V T; Levitus, Marcia; Wachter, Rebekka M

    2014-11-25

    We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

  18. Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle.

    PubMed

    Griffiths, Howard; Cousins, Asaph B; Badger, Murray R; von Caemmerer, Susanne

    2007-02-01

    A model defining carbon isotope discrimination (delta13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online delta13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of delta13C were higher than predicted from the ratio of intercellular to external CO2 (p(i)/p(a)) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g(i), was 0.044 mol m(-2) s(-1) bar(-1). A higher estimate of g(i) (0.085 mol m(-2) s(-1) bar(-1)) was needed to account for the modeled and measured delta18O discrimination throughout the dark period. The differences in estimates of g(i) from the two isotope measurements, and an offset of -5.5 per thousand between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase delta13C discrimination during nighttime CO2 fixation while reducing delta13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.

  19. The non-photosynthetic phosphoenolpyruvate carboxylases of the C4 dicot Flaveria trinervia -- implications for the evolution of C4 photosynthesis.

    PubMed

    Bläsing, Oliver E; Ernst, Karin; Streubel, Monika; Westhoff, Peter; Svensson, Per

    2002-07-01

    C4 phospho enolpyruvate carboxylases (PEPCase; EC 4.1.1.3) have evolved from ancestral non-photosynthetic (C3) isoforms during the evolution of angiosperms and thereby gained distinct kinetic and regulatory properties. In order to obtain insight into this evolutionary process we have studied the C3 isoforms, ppcB and ppcC, of the C4 dicot Flaveria trinervia (Spreng.) C. Mohr and compared them with the C4 enzyme of this species, ppcA, and its orthologue in the C3 species F. pringlei Gandoger. Phylogenetic analyses indicate that the ppcB PEPCase is the closest relative of the ppcA enzyme. In addition, the presence of ppcB also in the closely related C3 species F. pringlei suggests that this gene was present already in the ancestral C3 species and consequently that ppcA has evolved by gene duplication of ppcB. Investigation of the enzymatic properties of the ppcB and ppcC enzymes showed low and similar K(0.5)-PEP values and limited activation by glucose-6-phosphate, typical of non-photosynthetic PEPCases, at pH 8.0. However, at the more physiological pH of 7.6, the ppcC enzyme displayed a substantially higher K(0.5)-PEP than the ppcB counterpart, indicating their involvement in different metabolic pathways. This indication was strengthened by malate inhibition studies in which the ppcC enzyme showed 10 times higher tolerance to the inhibitor. The ppcA enzyme was, however, by far the most tolerant enzyme towards malate. Interestingly, the increased malate tolerance was correlated with a decrease in enzyme efficiency displayed by the turnover constant k(cat). We therefore suggest that the increased malate tolerance, which is imperative for an efficient C4 cycle, is connected with a decreased enzyme efficiency that in turn is compensated by increased enzyme expression.

  20. Photoaffinity labeling of ribulose-1,5-bisphosphate carboxylase/oxygenase activase with ATP gamma-benzophenone. Identification of the ATP gamma-phosphate binding domain.

    PubMed

    Salvucci, M E; Rajagopalan, K; Sievert, G; Haley, B E; Watt, D S

    1993-07-05

    The phosphate-binding domain of the ATP-binding site of tobacco Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activase was elucidated by photo-affinity labeling with a monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]ATP gamma BP). Covalent incorporation of [gamma-32P]ATP gamma BP into the 42-kDa Rubisco activase subunit was dependent upon irradiation with ultraviolet light. Photolabelling of Rubisco activase with ATP gamma BP exhibited saturation kinetics; the apparent Kd for photolabeling was 5 microM. Two lines of evidence showed that ATP gamma BP modified Rubisco activase at the ATP-binding domain. First, physiological concentrations of ATP and ADP afforded complete protection against photolabeling of Rubisco activase by ATP gamma BP. Second, photolysis of Rubisco activase in the presence of ATP gamma BP decreased both the ATPase and the Rubisco activating activities. Inactivation of enzyme activity was dependent on ATP gamma BP concentration and could be prevented by including ADP during photolabeling. The region of Rubisco activase that was modified by ATP gamma BP was identified by isolating photolabeled peptides. Sequence analysis showed that ATP gamma BP modified Rubisco activase in two distinct regions; one region, S117-A136, is adjacent to the P-loop and the other region, V223-T234, exhibits homology to a region of adenylate kinase that ligates the essential metal ion. Photolabeling of these two regions of Rubisco activase was consistent with modification of the ATP gamma-phosphate-binding domain of Rubisco activase with ATP gamma BP.

  1. A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

    PubMed

    Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

    1996-04-30

    Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation.

  2. Lysine residues involved in the hysteresis and in the regulatory sites of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase.

    PubMed

    Yokota, A; Tokai, H

    1993-11-01

    Lysine residues have been suggested to be involved in the hysteretic decrease of the activity of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and the binding of ribulose 1,5-bisphosphate to its regulatory sites [Yokota, A. & Tsujimoto, N. (1992) Eur. J. Biochem. 204, 901-909]. This work identifies the lysine residues and investigates the effects of their chemical modification on the course of RuBisCO reaction. The carbamylated form of RuBisCO reacted with trinitrobenzene sulfonate in three phases; an initial rapid, second slow, and final non-specific reaction. Lys-334 in loop 6, Lys-21, and Lys-128, all from the large subunits, were trinitrophenylated in the first 60-min reaction. Lys-305 of the large subunits was labeled in the next step. The modification of these residues was strongly suppressed in the enzyme form that had undergone hysteretic conformational change after binding 2-carboxyarabinitol 1,5-bisphosphate at its catalytic sites. Instead, Lys-450 of the large subunits and Lys-71 from the small subunits were newly modified in the quaternary complex. A higher concentration of 2-carboxyarabinitol 1,5-bisphosphate reduced the trinitrophenylation of the two residues to half. The modification of the carbamylated form of the enzyme for 30 min was expected to arylate Lys-21, Lys-128, and Lys-334 at random, and the course of the reaction of the partially modified enzyme was expected to deviate from that of the unmodified enzyme. Experimental results showed that this was the case.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. CO2-responsive expression and gene organization of three ribulose-1,5-bisphosphate carboxylase/oxygenase enzymes and carboxysomes in Hydrogenovibrio marinus strain MH-110.

    PubMed

    Yoshizawa, Yoichi; Toyoda, Koichi; Arai, Hiroyuki; Ishii, Masaharu; Igarashi, Yasuo

    2004-09-01

    Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, fixes CO2 by the Calvin-Benson-Bassham cycle. Strain MH-110 possesses three different sets of genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO): CbbLS-1 and CbbLS-2, which belong to form I (L8S8), and CbbM, which belongs to form II (Lx). In this paper, we report that the genes for CbbLS-1 (cbbLS-1) and CbbM (cbbM) are both followed by the cbbQO genes and preceded by the cbbR genes encoding LysR-type regulators. In contrast, the gene for CbbLS-2 (cbbLS-2) is followed by genes encoding carboxysome shell peptides. We also characterized the three RubisCOs in vivo by examining their expression profiles in environments with different CO2 availabilities. Immunoblot analyses revealed that when strain MH-110 was cultivated in 15% CO2, only the form II RubisCO, CbbM, was expressed. When strain MH-110 was cultivated in 2% CO2, CbbLS-1 was expressed in addition to CbbM. In the 0.15% CO2 culture, the expression of CbbM decreased and that of CbbLS-1 disappeared, and CbbLS-2 was expressed. In the atmospheric CO2 concentration of approximately 0.03%, all three RubisCOs were expressed. Transcriptional analyses of mRNA by reverse transcription-PCR showed that the regulation was at the transcriptional level. Electron microscopic observation of MH-110 cells revealed the formation of carboxysomes in the 0.15% CO2 concentration. The results obtained here indicate that strain MH-110 adapts well to various CO2 concentrations by using different types of RubisCO enzymes.

  4. Phylogeny and functional expression of ribulose 1,5-bisphosphate carboxylase/oxygenase from the autotrophic ammonia-oxidizing bacterium Nitrosospira sp. isolate 40KI.

    PubMed

    Utåker, Janne B; Andersen, Kjell; Aakra, Agot; Moen, Birgitte; Nes, Ingolf F

    2002-01-01

    The autotrophic ammonia-oxidizing bacteria (AOB), which play an important role in the global nitrogen cycle, assimilate CO(2) by using ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we describe the first detailed study of RubisCO (cbb) genes and proteins from the AOB. The cbbLS genes from Nitrosospira sp. isolate 40KI were cloned and sequenced. Partial sequences of the RubisCO large subunit (CbbL) from 13 other AOB belonging to the beta and gamma subgroups of the class Proteobacteria are also presented. All except one of the beta-subgroup AOB possessed a red-like type I RubisCO with high sequence similarity to the Ralstonia eutropha enzyme. All of these new red-like RubisCOs had a unique six-amino-acid insert in CbbL. Two of the AOB, Nitrosococcus halophilus Nc4 and Nitrosomonas europaea Nm50, had a green-like RubisCO. With one exception, the phylogeny of the AOB CbbL was very similar to that of the 16S rRNA gene. The presence of a green-like RubisCO in N. europaea was surprising, as all of the other beta-subgroup AOB had red-like RubisCOs. The green-like enzyme of N. europaea Nm50 was probably acquired by horizontal gene transfer. Functional expression of Nitrosospira sp. isolate 40KI RubisCO in the chemoautotrophic host R. eutropha was demonstrated. Use of an expression vector harboring the R. eutropha cbb control region allowed regulated expression of Nitrosospira sp. isolate 40KI RubisCO in an R. eutropha cbb deletion strain. The Nitrosospira RubisCO supported autotrophic growth of R. eutropha with a doubling time of 4.6 h. This expression system may allow further functional analysis of AOB cbb genes.

  5. Analysis of facultative lithotroph distribution and diversity on volcanic deposits by use of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase.

    PubMed

    Nanba, K; King, G M; Dunfield, K

    2004-04-01

    A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.

  6. Ribulose-1,5-bisphosphate carboxylase/oxygenase genes as a functional marker for chemolithoautotrophic halophilic sulfur-oxidizing bacteria in hypersaline habitats.

    PubMed

    Tourova, Tatjana P; Kovaleva, Olga L; Sorokin, Dimitry Yu; Muyzer, Gerard

    2010-07-01

    The presence and diversity of the cbb genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (a key enzyme of the Calvin-Benson cycle of autotrophic CO(2) assimilation) were investigated in pure cultures of seven genera of halophilic chemolithoautotrophic sulfur-oxidizing bacteria (SOB) and in sediments from a hypersaline lake in which such bacteria have been recently discovered. All of the halophilic SOB strains (with the exception of Thiohalomonas nitratireducens) possessed the cbbL gene encoding RuBisCO form I, while the cbbM gene encoding RuBisCO form II was detected only in some of the pure cultures. The general topologies of the CbbL/CbbM trees and the 16S rRNA gene tree were different, but both markers showed that the halophilic SOB genera formed independent lineages in the Gammaproteobacteria. In some cases, such as with several strains of the genus Thiohalospira and with Thioalkalibacter halophilus, the cbbL clustering was incongruent with the positions of these strains on the ribosomal tree. In the cbbM tree, the clustering of Thiohalospira and Thiohalorhabdus strains was incongruent with their branching in both cbbL and 16S rRNA gene trees. cbbL and cbbM genes related to those found in the analysed halophilic SOB were also detected in a sediment from a hypersaline lake in Kulunda Steppe (Russia). Most of the cbbL and cbbM genes belonged to members of the genus Thiohalorhabdus. In the cbbL clone library, sequences related to those of Halothiobacillus and Thiohalospira were detected as minor components. Some of the environmental cbbM sequences belonged to as yet unknown phylotypes, representing deep lineages of halophilic autotrophs.

  7. Deduced amino acid sequence, functional expression, and unique enzymatic properties of the form I and form II ribulose bisphosphate carboxylase/oxygenase from the chemoautotrophic bacterium Thiobacillus denitrificans.

    PubMed

    Hernandez, J M; Baker, S H; Lorbach, S C; Shively, J M; Tabita, F R

    1996-01-01

    The cbbL cbbS and cbbM genes of Thiobacillus denitrificans, encoding form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), respectively, were found to complement a RubisCO-negative mutant of Rhodobacter sphaeroides to autotrophic growth. Endogenous T. denitrificans promoters were shown to function in R. sphaeroides, resulting in high levels of cbbL cbbS and cbbM expression in the R. sphaeroides host. This expression system provided high levels of both T. denitrificans enzymes, each of which was highly purified. The deduced amino acid sequence of the form I enzyme indicated that the large subunit was closely homologous to previously sequenced form I RubisCO enzymes from sulfur-oxidizing bacteria. The form I T. denitrificans enzyme possessed a very low substrate specificity factor and did not exhibit fallover, and yet this enzyme showed a poor ability to recover from incubation with ribulose 1,5-bisphosphate. The deduced amino acid sequence of the form II T. denitrificans enzyme resembled those of other form II RubisCO enzymes. The substrate specificity factor was characteristically low, and the lack of fallover and the inhibition by ribulose 1,5-bisphosphate were similar to those of form II RubisCO obtained from nonsulfur purple bacteria. Both form I and form II RubisCO from T. denitrificans possessed high KCO2 values, suggesting that this organism might suffer in environments containing low levels of dissolved CO2. These studies present the initial description of the kinetic properties of form I and form II RubisCO from a chemoautotrophic bacterium that synthesizes both types of enzyme.

  8. Analysis of Facultative Lithotroph Distribution and Diversity on Volcanic Deposits by Use of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase†

    PubMed Central

    Nanba, K.; King, G. M.; Dunfield, K.

    2004-01-01

    A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass. PMID:15066819

  9. Expressed genes for plant-type ribulose 1,5-bisphosphate carboxylase/oxygenase in the photosynthetic bacterium Chromatium vinosum, which possesses two complete sets of the genes.

    PubMed Central

    Viale, A M; Kobayashi, H; Akazawa, T

    1989-01-01

    Two sets of genes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were detected in the photosynthetic purple sulfur bacterium Chromatium vinosum by hybridization analysis with RuBisCO gene probes, cloned by using the lambda Fix vector, and designated rbcL-rbcS and rbcA-rbcB. rbcL and rbcA encode the large subunits, and rbcS and rbcB encode the small subunits. rbcL-rbcS was the same as that reported previously (A. M. Viale, H. Kobayashi, T. Takabe, and T. Akazawa, FEBS Lett. 192:283-288, 1985). A DNA fragment bearing rbcA-rbcB was subcloned in plasmid vectors and sequenced. We found that rbcB was located 177 base pairs downstream of the rbcA coding region, and both genes were preceded by plausible procaryotic ribosome-binding sites. rbcA and rbcD encoded polypeptides of 472 and 118 amino acids, respectively. Edman degradation analysis of the subunits of RuBisCO isolated from C. vinosum showed that rbcA-rbcB encoded the enzyme present in this bacterium. The large- and small-subunit polypeptides were posttranslationally processed to remove 2 and 1 amino acid residues from their N-termini, respectively. Among hetero-oligomeric RuBisCOs, the C. vinosum large subunit exhibited higher homology to that from cyanobacteria, eucaryotic algae, and higher plants (71.6 to 74.2%) than to that from the chemolithotrophic bacterium Alcaligenes eutrophus (56.6%). A similar situation has been observed for the C. vinosum small subunit, although the homology among small subunits from different organisms was lower than that among the large subunits. Images PMID:2708310

  10. Multiple E-Boxes in the Distal Promoter of the Rat Pyruvate Carboxylase Gene Function as a Glucose-Responsive Element

    PubMed Central

    Muangsawat, Sureeporn; Boonsaen, Thirajit; MacDonald, Michael J.; Jitrapakdee, Sarawut

    2014-01-01

    Pyruvate carboxylase (PC) is an anaplerotic enzyme that regulates glucose-induced insulin secretion in pancreatic islets. Dysregulation of its expression is associated with type 2 diabetes. Herein we describe the molecular mechanism underlying the glucose-mediated transcriptional regulation of the PC gene. Incubation of the rat insulin cell line INS-1 832/13 with glucose resulted in a 2-fold increase in PC mRNA expression. Transient transfections of the rat PC promoter-luciferase reporter construct in the above cell line combined with mutational analysis indicated that the rat PC gene promoter contains the glucose-responsive element (GRE), comprising three canonical E-boxes (E1, E3 and E4) and one E-box-like element (E2) clustering between nucleotides –546 and –399, upstream of the transcription start site. Mutation of any of these E-boxes resulted in a marked reduction of glucose-mediated transcriptional induction of the reporter gene. Electrophoretic mobility shift assays revealed that the upstream stimulatory factors 1 and 2 (USF1 and USF2) bind to E1, the Specificity Protein-1 (Sp1) binds to E2, USF2 and the carbohydrate responsive element binding protein (ChREBP) binds to E4, while unknown factors binds to E3. High glucose promotes the recruitment of Sp1 to E2 and, USF2 and ChREBP to E4. Silencing the expression of Sp1, USF2 and ChREBP by their respective siRNAs in INS-1 832/13 cells blunted glucose-induced expression of endogenous PC. We conclude that the glucose-mediated transcriptional activation of the rat PC gene is regulated by at least these three transcription factors. PMID:25054881

  11. Enhanced drought tolerance in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene via NO and Ca(2+).

    PubMed

    Qian, Baoyun; Li, Xia; Liu, Xiaolong; Chen, Pingbo; Ren, Chengang; Dai, Chuanchao

    2015-03-01

    We determined the effects of endogenous nitric oxide and Ca(2+) on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) under drought. In this study, seedlings were subjected to PEG 6000 treatments using PC and wild type (WT; Kitaake). The results showed that, compared with WT, PC had higher relative water content (RWC) and net photosynthetic rate (Pn) under drought. During a 2-day re-watering treatment, Pn recovered faster in PC than in WT. Further analyses showed that, under the drought treatment, the amount of endogenous hydrogen peroxide (H2O2) increased in WT mainly via NADPH oxidase. While in PC, the endogenous nitric oxide (NO) content increased via nitrate reductase and nitric oxide synthase on day 2 of the drought treatment and day 1 of the re-watering treatment. After 2 days of drought treatment, PC also showed higher PEPC activity, calcium content, phospholipase D (PLD) activity, C4-pepc and NAC6 transcript levels, and protein kinase activity as compared with PC without treatment. These changes did not occur in WT. Correlation analysis also proved NO associated with these indicators in PC. Based on these results, there was a particular molecular mechanism of drought tolerance in PC. The mechanism is related to the signaling processes via NO and Ca(2+) involving the protein kinase and the transcription factor, resulted in up-regulation of PEPC activity and its gene expression, such as C4pepc. Some genes encode antioxidant system, cu/zn-sod as well, which promote antioxidant system to clear MDA and superoxide anion radical, thereby conferring drought tolerance.

  12. Phylogeny and Functional Expression of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Autotrophic Ammonia-Oxidizing Bacterium Nitrosospira sp.Isolate 40KI

    PubMed Central

    Utåker, Janne B.; Andersen, Kjell; Aakra, Ågot; Moen, Birgitte; Nes, Ingolf F.

    2002-01-01

    The autotrophic ammonia-oxidizing bacteria (AOB), which play an important role in the global nitrogen cycle, assimilate CO2 by using ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we describe the first detailed study of RubisCO (cbb) genes and proteins from the AOB. The cbbLS genes from Nitrosospira sp. isolate 40KI were cloned and sequenced. Partial sequences of the RubisCO large subunit (CbbL) from 13 other AOB belonging to the β and γ subgroups of the class Proteobacteria are also presented. All except one of the β-subgroup AOB possessed a red-like type I RubisCO with high sequence similarity to the Ralstonia eutropha enzyme. All of these new red-like RubisCOs had a unique six-amino-acid insert in CbbL. Two of the AOB, Nitrosococcus halophilus Nc4 and Nitrosomonas europaea Nm50, had a green-like RubisCO. With one exception, the phylogeny of the AOB CbbL was very similar to that of the 16S rRNA gene. The presence of a green-like RubisCO in N. europaea was surprising, as all of the other β-subgroup AOB had red-like RubisCOs. The green-like enzyme of N. europaea Nm50 was probably acquired by horizontal gene transfer. Functional expression of Nitrosospira sp. isolate 40KI RubisCO in the chemoautotrophic host R. eutropha was demonstrated. Use of an expression vector harboring the R. eutropha cbb control region allowed regulated expression of Nitrosospira sp. isolate 40KI RubisCO in an R. eutropha cbb deletion strain. The Nitrosospira RubisCO supported autotrophic growth of R. eutropha with a doubling time of 4.6 h. This expression system may allow further functional analysis of AOB cbb genes. PMID:11751824

  13. 1α,25-dihydroxyvitamin D inhibits de novo fatty acid synthesis and lipid accumulation in metastatic breast cancer cells through down-regulation of pyruvate carboxylase.

    PubMed

    Wilmanski, Tomasz; Buhman, Kimberly; Donkin, Shawn S; Burgess, John R; Teegarden, Dorothy

    2017-02-01

    Both increased de novo fatty acid synthesis and higher neutral lipid accumulation are a common phenotype observed in aggressive breast cancer cells, making lipid metabolism a promising target for breast cancer prevention. In the present studies, we demonstrate a novel effect of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)₂D) on lipid metabolism in malignant breast epithelial cells. Treatment of MCF10CA1a breast epithelial cells with 1,25(OH)₂D (10 nM) for 5 and 7 days decreased the level of triacylglycerol, the most abundant form of neutral lipids, by 20%(±3.9) and 50%(±5.9), respectively. In addition, 1,25(OH)₂D treatment for 5 days decreased palmitate synthesis from glucose, the major fatty acid synthesized de novo (48%±5.5 relative to vehicle). We have further identified the anaplerotic enzyme pyruvate carboxylase (PC) as a target of 1,25(OH)₂D-mediated regulation and hypothesized that 1,25(OH)₂D regulates breast cancer cell lipid metabolism through inhibition of PC. PC mRNA expression was down-regulated with 1,25(OH)₂D treatment at 2 (73%±6 relative to vehicle) and 5 (56%±8 relative to vehicle) days. Decrease in mRNA abundance corresponded with a decrease in PC protein expression at 5 days of treatment (54%±12 relative to vehicle). Constitutive overexpression of PC in MCF10CA1a cells using a pCMV6-PC plasmid inhibited the effect of 1,25(OH)₂D on both TAG accumulation and de novo palmitate synthesis from glucose. Together, these studies demonstrate a novel mechanism through which 1,25(OH)₂D regulates lipid metabolism in malignant breast epithelial cells.

  14. Pyramiding expression of maize genes encoding phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) synergistically improve the photosynthetic characteristics of transgenic wheat.

    PubMed

    Zhang, HuiFang; Xu, WeiGang; Wang, HuiWei; Hu, Lin; Li, Yan; Qi, XueLi; Zhang, Lei; Li, ChunXin; Hua, Xia

    2014-09-01

    Using particle bombardment transformation, we introduced maize pepc cDNA encoding phosphoenolpyruvate carboxylase (PEPC) and ppdk cDNA encoding pyruvate orthophosphate dikinase (PPDK) into the C3 crop wheat to generate transgenic wheat lines carrying cDNA of pepc (PC lines), ppdk (PK lines) or both (PKC lines). The integration, transcription, and expression of the foreign genes were confirmed by Southern blot, Real-time quantitative reverse transcription PCR (Q-RT-PCR), and Western blot analysis. Q-RT-PCR results indicated that the average relative expression levels of pepc and ppdk in the PKC lines reached 10 and 4.6, respectively, compared to their expressions in untransformed plants (set to 1). The enzyme activities of PEPC and PPDK in the PKC lines were 4.3- and 2.1-fold higher, respectively, than in the untransformed control. The maximum daily net photosynthetic rates of the PKC, PC, and PK lines were enhanced by 26.4, 13.3, and 4.5%, respectively, whereas the diurnal accumulations of photosynthesis were 21.3, 13.9, and 6.9%, respectively, higher than in the control. The Fv/Fm of the transgenic plants decreased less than in the control under high temperature and high light conditions (2 weeks after anthesis), suggesting that the transgenic wheat transports more absorbed light energy into a photochemical reaction. The exogenous maize C4-specific pepc gene was more effective than ppdk at i