RESUMO
Rectangular V-amylose single crystals were prepared by adding racemic ibuprofen to hot dilute aqueous solutions of native and enzymatically-synthesized amylose. The lamellar thickness increased with increasing degree of polymerization of amylose and reached a plateau at about 7 nm, consistent with a chain-folding mechanism. The CP/MAS NMR spectrum as well as base-plane electron and powder X-ray diffraction patterns recorded from hydrated specimens were similar to those of V-amylose complexed with propan-2-ol. Amylose was crystallized in an orthorhombic unit cell with parameters a = 2.824 ± 0.001 nm, b = 2.966 ± 0.001 nm, and c = 0.800 ± 0.001 nm. A molecular model was proposed based on structural analogies with the Vpropan-2-ol complex and on assumptions on the stoichiometry of ibuprofen. The unit cell would contain four antiparallel 7-fold amylose single helices with ibuprofen molecules distributed inside and between the helices.
Assuntos
Amilose/química , Ibuprofeno/química , Nanopartículas/química , Varredura Diferencial de Calorimetria , Cristalização , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Estrutura Molecular , Nanoconjugados/química , Polimerização , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
In vitro polymerization of ß-mannans is a challenging reaction due to the steric hindrance confered by the configuration of mannosyl residues and the thermodynamic instability of the ß-anomer. Whatever the approach used to date-whether chemical, or enzymatic with glycosynthases and mannosyltransferases-pure ß-1,4-mannans have never been synthesized in vitro. This has limited attempts to investigate their role in the production of plant and algal cell walls, in which they are highly abundant. It has also impeded the exploitation of their properties as biosourced materials. In this paper, we demonstrate that TM1225, a thermoactive glycoside phosphorylase from the hyperthermophile species Thermotoga maritima, is a powerful biocatalytic tool for the ecofriendly synthesis of pure ß-1,4-mannan. The recombinant production of this enzyme and its biochemical characterization allowed us to prove that it catalyzes the reversible phosphorolysis of ß-1,4-mannosides, and determine its role in the metabolism of the algal mannans on which T. maritima feeds in submarine sediments. Furthermore, after optimizing the reaction conditions, we exploited the synthetic ability of TM1225 to produce ß-1,4-mannan in vitro. At 60 °C and from d-mannose 1-phosphate and mannohexaose, the enzyme synthesized mannoside chains with a degree of polymerization up to 16, which precipitated into lamellar single crystals. The X-ray powder diffraction and base-plane electron diffraction patterns of the lamellar crystals unambiguously show that the synthesized product belongs to the mannan I family previously observed in planta in pure linear mannans, such as those of the ivory nut. The in vitro formation of these mannan I crystals is likely determined by the high reaction temperature and the narrow chain length distribution of the insoluble chains.
Assuntos
Biocatálise , Mananas/síntese química , Proteínas de Bactérias/metabolismo , Cristalização , Fosforilases/metabolismo , Polimerização , Thermotoga maritima/enzimologiaRESUMO
A gene encoding a novel dextransucrase was identified in the genome of Oenococcus kitaharae DSM17330 and cloned into E. coli. With a kcat of 691s-1 and a half-life time of 111h at 30°C, the resulting recombinant enzyme -named DSR-OK- stands as one of the most efficient and stable dextransucrase characterized to date. From sucrose, this enzyme catalyzes the synthesis of a quasi linear dextran with a molar mass higher than 1×109g·mol-1 that presents uncommon rheological properties such as a higher viscosity than that of the most industrially used dextran from L. mesenteroides NRRL-B-512F, a yield stress that was never described before for any type of dextran, as well as a gel-like structure. All these properties open the way to a vast array of new applications in health, food/feed, bulk or fine chemicals fields.
Assuntos
Dextranos/biossíntese , Glucosiltransferases/metabolismo , Oenococcus/enzimologia , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Biocatálise , Configuração de Carboidratos , Bases de Dados de Proteínas , Glucosiltransferases/genética , Oenococcus/genética , Proteínas Recombinantes/genética , Sacarose/química , Sacarose/metabolismo , ViscosidadeRESUMO
New α(1â2) or α(1â3) branched dextrans with high molar masses and controlled architecture were synthesized using a dextransucrase and branching sucrases. Their molecular structure, solubility, conformation, film-forming ability, as well as their thermal and mechanical properties were determined. These new dextrans present structures with low densities from 9,500 to 14,000gm-3 in H2O/DMSO medium, their molar mass, size and dispersity increase with increasing branching degree (weight-average molar mass up to 109gmol-1 and radius of gyration around 500nm). Dextrans exhibit a glass transition between 40.5 and 63.2°C for water content varying from 12.2 to 14.1%. The effect of branching is mainly observed on the ability of dextran to crystallize. They have a good film-forming ability with a storage modulus which varies from 2 to 4GPa within a relative humidity range of 10-50%.
Assuntos
Dextranos/química , Dextranos/metabolismo , Glucosiltransferases/metabolismo , Substâncias Macromoleculares/química , Sacarase/metabolismo , Varredura Diferencial de Calorimetria , Dextranos/ultraestrutura , Módulo de Elasticidade , Vidro , Umidade , Hidrodinâmica , Peso Molecular , Sacarose/metabolismo , Temperatura de Transição , Água/químicaRESUMO
Asymmetrical flow field flow fractionation (AF4) has proven to be a very powerful and quantitative method for the determination of the macromolecular structure of high molar mass branched biopolymers, when coupled with multi-angle laser light scattering (MALLS). This work describes a detailed investigation of the macromolecular structure of native glycogens and hyperbranched α-glucans (HBPs), with average molar mass ranging from 2 × 10(6) to 4.3 × 10(7) g mol(-1), which are not well fractionated by means of classical size-exclusion chromatography. HBPs were enzymatically produced from sucrose by the tandem action of an amylosucrase and a branching enzyme mimicking in vitro the elongation and branching steps involved in glycogen biosynthesis. Size and molar mass distributions were studied by AF4, coupled with online quasi-elastic light scattering (QELS) and transmission electron microscopy. AF4-MALLS-QELS has shown a remarkable agreement between hydrodynamic radii obtained by online QELS and by AF4 theory in normal mode with constant cross flow. Molar mass, size, and dispersity were shown to significantly increase with initial sucrose concentration, and to decrease when the branching enzyme activity increases. Several populations with different size range were observed: the amount of small size molecules decreasing with increasing sucrose concentration. The spherical and dense global conformation thus highlighted was partly similar to native glycogens. A more detailed study of HBPs synthesized from low and high initial sucrose concentrations was performed using complementary enzymatic hydrolysis of external chains and chromatography. It emphasized a more homogeneous branching pattern than native glycogens with a denser core and shorter external chains.
Assuntos
Fracionamento por Campo e Fluxo , Glucanos/química , Glicogênio/química , Amilases/metabolismo , Bactérias/enzimologia , Fracionamento por Campo e Fluxo/métodos , Glucanos/isolamento & purificação , Glucanos/metabolismo , Glucosiltransferases/metabolismo , Glicogênio/isolamento & purificação , Glicogênio/metabolismo , Luz , Estrutura Molecular , Peso Molecular , Espalhamento de Radiação , Sacarose/metabolismoRESUMO
Pea (Pisum sativum L.) productivity is linked to its ability to cope with abiotic stresses such as low temperatures during fall and winter. In this study, we investigate the chloroplast-related changes occurring during pea cold acclimation, in order to further lead to genetic improvement of its field performance. Champagne and Térèse, two pea lines with different acclimation capabilities, were studied by physiological measurements, sub-cellular fractionation followed by relative protein quantification and two-dimensional DIGE. The chilling tolerance might be related to an increase in protein related to soluble sugar synthesis, antioxidant potential, regulation of mRNA transcription and translation through the chloroplast. Freezing tolerance, only observed in Champagne, seems to rely on a higher inherent photosynthetic potential at the beginning of the cold exposure, combined with an early ability to start metabolic processes aimed at maintaining the photosynthetic capacity, optimizing the stoichiometry of the photosystems and inducing dynamic changes in carbohydrate and protein synthesis and/or turnover.
Assuntos
Aclimatação , Cloroplastos/química , Temperatura Baixa , Pisum sativum/fisiologia , Biomassa , Carbono/química , Clorofila/química , Cloroplastos/genética , Transporte de Elétrons , Eletroforese em Gel Bidimensional , Congelamento , Regulação da Expressão Gênica de Plantas , Genótipo , Nitrogênio/química , Estresse Oxidativo , Pisum sativum/metabolismo , Fotoquímica , Fotossíntese , Complexo de Proteína do Fotossistema II , Proteínas de Plantas/metabolismo , Proteoma , Proteômica , RNA Mensageiro/metabolismo , Transdução de Sinais , Frações SubcelularesRESUMO
Glycogen biosynthesis requires the coordinated action of elongating and branching enzymes, of which the synergetic action is still not clearly understood. We have designed an experimental plan to develop and fully exploit a biomimetic system reproducing in vitro the activities involved in the formation of α(1,4) and α(1,6) glycosidic linkages during glycogen biosynthesis. This method is based on the use of two bacterial transglucosidases, the amylosucrase from Neisseria polysaccharea and the branching enzyme from Rhodothermus obamensis . The α-glucans synthesized from sucrose, a low cost agroresource, by the tandem action of the two enzymes, have been characterized by using complementary enzymatic, chromatographic, and imaging techniques. In a single step, linear and branched α-glucans were obtained, whose proportions, morphology, molar mass, and branching degree depended on both the initial sucrose concentration and the ratio between elongating and branching enzymes. In particular, spherical hyperbranched α-glucans with a controlled mean diameter (ranging from 10 to 150 nm), branching degree (from 10 to 13%), and weight-average molar mass (3.7 × 10(6) to 4.4 × 10(7) g.mol(-1)) were synthesized. Despite their structure, which is similar to that of natural glycogens, the mechanisms involved in their in vitro synthesis appeared to be different from those involved in the biosynthesis of native hyperbranched α-glucans.
Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Glucanos/síntese química , Glucosiltransferases/metabolismo , Neisseria/enzimologia , Rhodothermus/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Biomimética , Glucanos/química , Glucanos/ultraestrutura , Glucosiltransferases/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Amido/metabolismoRESUMO
BACKGROUND: Glycogen and starch branching enzymes catalyze the formation of α(1â6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1â4) linkage and transfer in α(1â6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch. METHODS: The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure. RESULTS: A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3-8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30-70nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family. CONCLUSIONS: RoBE is able to transfer α(1â4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1â4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family. GENERAL SIGNIFICANCE: This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro.
Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Rhodothermus/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Proteínas de Bactérias/metabolismo , Catálise , Estabilidade Enzimática , Especificidade por Substrato/fisiologiaRESUMO
Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds.
Assuntos
Carbono/metabolismo , Redes e Vias Metabólicas , Modelos Biológicos , Proteínas de Plantas/metabolismo , Plastídeos/enzimologia , Amido/biossíntese , Zea mays/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Sequência de Aminoácidos , Cromatografia de Afinidade , Cromatografia em Gel , Glucanos/metabolismo , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Imunoprecipitação , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/isolamento & purificação , Extratos Vegetais , Proteínas de Plantas/química , Ligação Proteica , Estrutura Terciária de Proteína , Piruvato Ortofosfato Diquinase/química , Piruvato Ortofosfato Diquinase/metabolismo , Sintase do Amido/química , Sintase do Amido/metabolismoRESUMO
In addition to the exclusively granule-bound starch synthase GBSSI, starch granules also bind significant proportions of other starch biosynthetic enzymes, particularly starch synthases (SS) SSI and SSIIa, and starch branching enzyme (BE) BEIIb. Whether this association is a functional aspect of starch biosynthesis, or results from non-specific entrapment during amylopectin crystallization, is not known. This study utilized genetic, immunological, and proteomic approaches to investigate comprehensively the proteome and phosphoproteome of Zea mays endosperm starch granules. SSIII, BEI, BEIIa, and starch phosphorylase were identified as internal granule-associated proteins in maize endosperm, along with the previously identified proteins GBSS, SSI, SSIIa, and BEIIb. Genetic analyses revealed three instances in which granule association of one protein is affected by the absence of another biosynthetic enzyme. First, eliminating SSIIa caused reduced granule association of SSI and BEIIb, without affecting GBSS abundance. Second, eliminating SSIII caused the appearance of two distinct electrophoretic mobility forms of BEIIb, whereas only a single migration form of BEIIb was observed in wild type or any other mutant granules examined. Third, eliminating BEIIb caused significant increases in the abundance of BEI, BEIIa, SSIII, and starch phosphorylase in the granule, without affecting SSI or SSIIa. Analysis of the granule phosphoproteome with a phosphorylation-specific dye indicated that GBSS, BEIIb, and starch phosphorylase are all phosphorylated as they occur in the granule. These results suggest the possibility that starch metabolic enzymes located in granules are regulated by post-translational modification and/or protein-protein interactions.