RESUMO
Engineering glycoside hydrolases is a major route to obtaining catalysts forming glycosidic bonds. Glycosynthases, thioglycoligases, and transglycosylases represent the main strategies, each having advantages and drawbacks. Here, we show that an engineered enzyme from the GH84 family, the acid-base mutant TtOGA-D120N, is an efficient O-, N-, and S-glycoligase, able to use Ssp3, Osp3, Nsp2, and Nsp nucleophiles. Moreover, TtOGA-D120N catalyzes the formation and release of N-acetyl-d-glucosamine 1,2-oxazoline, the intermediate of hexosaminidases displaying substrate-assisted catalysis. This release of an activated intermediate allows cascade synthesis by combination with transglycosylases or glycosynthases, here exemplified by synthesis of the human milk oligosaccharide lacto-N-triose II.
Assuntos
Hexosaminidases , beta-N-Acetil-Hexosaminidases , Humanos , Glicosilação , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/metabolismo , Glicosiltransferases , Glicosídeo Hidrolases/metabolismoRESUMO
Family 1 glycosyltransferases (GT1s, UGTs) catalyze the regioselective glycosylation of natural products in a single step. We identified GmUGT88E3 as a particularly promising biocatalyst able to produce a variety of pure, single glycosidic products from polyphenols with high chemical yields. We investigated this particularly desirable duality toward specificity, i.e., promiscuous toward acceptors while regiospecific. Using high-field NMR, kinetic characterization, molecular dynamics simulations, and mutagenesis studies, we uncovered that the main molecular determinant of GmUGT88E3 specificity is a methionine-aromatic bridge, an interaction often present in protein structures but never reported for enzyme-substrate interactions. Here, mutating Met127 led to inactive proteins or 100-fold reduced activity.
Assuntos
Glycine max , Glicosiltransferases , Glicosiltransferases/metabolismo , Glycine max/genética , Metionina/metabolismo , Glicosilação , Glicosídeos , Racemetionina/metabolismo , Especificidade por SubstratoRESUMO
Pneumococcal capsules are important in pneumococcal pathogenesis and vaccine development. Although conjugate vaccines have brought about a significant reduction in invasive pneumococcal disease (IPD) caused by vaccine serotypes, the relative serotype prevalence has shifted with the dramatic emergence of serotype 24F in some countries. Here, we describe 14 isolates (13 IPD and 1 non-IPD) expressing a new capsule type, 24C, which resembles 24F but has a novel serological profile. We also describe the antigenic, biochemical, and genetic basis of 24F and 24C and the related serotypes 24A and 24B. Structural studies show that 24B, 24C, and 24F have identical polysaccharide backbones [ß-Ribf-(1â4)-α-Rhap-(1â3)-ß-GlcpNAc-(1â4)-ß-Rhap-(1â4)-ß-Glcp] but with different side chains, as follows: 24F has arabinitol-phosphate and 24B has ribitol-phosphate. 24C has a mixture of 24F and 24B repeating units, with the ratio of ribitol to arabinitol being strain dependent. In contrast, the 24A capsule has a backbone without ß-Ribf but with arabinitol-phosphate and phosphocholine side chains. These structures indicate that factor-sera 24d and 24e recognize arabinitol and ribitol, respectively, which explains the serology of serogroup 24, including those of 24C. The structures can be genetically described by the bispecificity of wcxG, which is capable of transferring arabinitol or ribitol when arabinitol is limiting. Arabinitol is likely not produced in 24B but is produced in reduced amounts in 24C due to various mutations in abpA or abpB genes. Our findings demonstrate how pneumococci modulate their capsule structure and immunologic properties with small genetic changes, thereby evading host immune responses. Our findings also suggest a potential for new capsule types within serogroup 24.
Assuntos
Infecções Pneumocócicas , Streptococcus pneumoniae , Humanos , Vacinas Pneumocócicas , Sorogrupo , Streptococcus pneumoniae/genética , Vacinas ConjugadasRESUMO
Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6-12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d/l-, α/ß-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.
Assuntos
Glicosídeo Hidrolases , Glicosiltransferases , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Glicosilação , Glicosiltransferases/genética , Hidrólise , Oligossacarídeos , Especificidade por SubstratoRESUMO
The transglycosylation abilities of ß-galactosidases were investigated using hyperpolarized [U-13C,U-2H]glucose as an acceptor and o-nitrophenyl ß-galactopyranoside as a donor. Several products were readily observable, and at least in the case when O3 acted as an acceptor, the enzymes showed a clear selectivity toward the ß-anomer of glucose. Additionally, it was possible to determine the relative hydrolysis rates of the formed transglycosylation products, providing information on the selectivity as well. Using this method, the transglycosylation abilities of the enzymes could be studied at a very high temporal resolution as well as with high sensitivity, and due to the relative ease of the setup, this method could be more generally applied to investigate glycosidases.
Assuntos
Ressonância Magnética Nuclear Biomolecular , beta-Galactosidase/metabolismo , Galactose/química , Galactose/metabolismo , Glucose/química , Glucose/metabolismo , Glicosilação , Cinética , Estereoisomerismo , Especificidade por SubstratoRESUMO
Capsular polysaccharides (CPS) are crucial virulence factors of Streptococcus pneumoniae The previously unknown CPS structures of the pneumococcal serogroup 16 (serotypes 16F and 16A) were thoroughly elucidated by nuclear magnetic resonance (NMR) spectroscopy and verified by chemical analysis. The following repeat unit structures were determined: 16F, -3)-α-l-Rhap-[4-P-1-Gro]-(1-3)-α-d-Glcp-[(6-P-1)-Gro]-(1-3)-ß-l-Rhap-[2-OAc]-(1-4)-ß-d-Glcp-(1-; 16A, -3)-ß-d-Galf-[2-OAc (70%)]-(1-3)-α-l-Rhap-(1-2)-α-l-Rhap-(1-3)-α-d-Galp-[(6-P-1)-Gro]-(1-3)-ß-d-Galp-(1-4)-ß-d-Glcp-(1- (OAc, O-acetyl substitution; P-1-Gro, glycerol-1-phosphate substitution) A further analysis of CPS biosynthesis of serotypes 16F and 16A, in conjunction with published cps gene bioinformatics analysis and structures of related serotypes, revealed presumable specific function of glycosyltransferase, acetyltransferase, phosphotransferase, and polymerase. The functions of glycosyltransferases WcxN and WcxT were proposed for the first time, and they were assigned to catalyze linkage of α-l-Rhap-(1-3)-α-d-Glcp and α-l-Rhap-(1-2)-α-l-Rhap, respectively. Furthermore, since serotype 16F was genetically close to serogroup 28, cross-reactions between serogroup 16 and serogroup 28 were studied using diagnostic antisera, which provided further understanding of antigenic properties of CPS and diagnostic antisera. Interestingly, serotype 16F cross-reacted with factor antisera 28b and 11c. Meanwhile, serotype 16A cross-reacted with factor antiserum 11c.IMPORTANCE The vaccine pressure against Streptococcus pneumoniae could result in a change of prevalence in carriage and invasive serotypes. As such, it is necessary to monitor the distribution to achieve successful vaccination of the population, and similarly, it is important to increase the knowledge of even the currently less prevalent serotypes. The CPS are vital for the virulence of the pathogen, and antigenic properties of CPS are based on the structure. Consequently, a better understanding of the structure, biosynthesis, and serology of the capsular polysaccharides can be of great importance toward developing future diagnostic tools and vaccines.
Assuntos
Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Polissacarídeos Bacterianos/química , Streptococcus pneumoniae/imunologia , Animais , Proteínas de Bactérias/metabolismo , Sequência de Carboidratos , Reações Cruzadas , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Soros Imunes/metabolismo , Espectroscopia de Ressonância Magnética , Mutação , Polissacarídeos Bacterianos/imunologia , Coelhos , Sorogrupo , Streptococcus pneumoniae/químicaRESUMO
Using dissolution dynamic nuclear polarization, the sensitivity of single scan solution state 13C NMR can be improved up to 4 orders of magnitude. In this study, the enzyme lacZ ß-galactosidase from Escherichia coli was subjected to hyperpolarized substrate, and previously unknown reaction intermediates were observed, including a 1,1-linked disaccharide. The enzyme is known for making 1,6-transglycosylation, producing products like allolactose, that are also substrates. To analyze the kinetics, a simple kinetic model was developed and used to determine relative transglycosylation and hydrolysis rates of each of the intermediates, and the novel transglycosylation intermediates were determined as better substrates than the 1,6-linked one, explaining their transient nature. These findings suggest that hydrolysis and transglycosylation might be more complex than previously described.
RESUMO
There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galß1-4GlcNAcß-tetramethylrhodamine (LacNAc-TMR (Galß1-4GlcNAcß(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcß1-4GlcNAcß-TMR), LacNAc-TMR, and LacNAcß1-6LacNAcß-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the ß1-6 bond in LacNAcß1-6LacNAcß-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis.
Assuntos
Proteínas de Bactérias/metabolismo , Quitinases/metabolismo , Proteínas de Insetos/metabolismo , Lactose/análogos & derivados , Salmonella typhimurium/enzimologia , Amino Açúcares/química , Amino Açúcares/metabolismo , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Sequência de Carboidratos , Quitina/química , Quitina/metabolismo , Quitinases/classificação , Quitinases/genética , Variação Genética , Humanos , Hidrólise , Proteínas de Insetos/genética , Insetos , Cinética , Lactose/química , Lactose/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Filogenia , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica , Rodaminas/química , Rodaminas/metabolismo , Salmonella typhimurium/genética , Especificidade por Substrato , VertebradosRESUMO
Regioselective glycosylation is a chemical challenge, leading to multistep syntheses with protecting group manipulations, ultimately resulting in poor atom economy and compromised sustainability. Enzymes allow eco-friendly and regioselective bond formation with fully deprotected substrates in a single reaction. For the selective glucosylation of silibinin, a pharmaceutical challenged with low solubility, enzyme engineering has previously been employed, but the resulting yields and k cat were limited, prohibiting the application of the engineered catalyst. Here, we identified a naturally regioselective silibinin glucosyltransferase, UGT706F8, a family 1 glycosyltransferase from Zea mays. It selectively and efficiently (k cat = 2.1 ± 0.1 s-1; K M = 36.9 ± 5.2 µM; TTN = 768 ± 22) catalyzes the quantitative synthesis of silibinin 7-O-ß-d-glucoside. We solved the crystal structure of UGT706F8 and investigated the molecular determinants of regioselective silibinin glucosylation. UGT706F8 was the only regioselective enzyme among 18 glycosyltransferases found to be active on silibinin. We found the temperature optimum of UGT706F8 to be 34 °C and the pH optimum to be 7-8. Our results indicate that UGT706F8 is an efficient silibinin glycosyltransferase that enables biocatalytic production of silbinin 7-O-ß-d-glucoside.
RESUMO
Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin-like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29-31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acid-long defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline-linked arabinogalactan chains with one galactose and 5 to 20 and more alpha-arabinofuranosyl residues with some beta-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked beta-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with >30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4.
Assuntos
Alérgenos/genética , Ambrosia/genética , Artemisia/genética , Proteínas de Plantas/genética , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Alérgenos/química , Alérgenos/imunologia , Alérgenos/isolamento & purificação , Ambrosia/química , Ambrosia/imunologia , Antígenos de Plantas , Artemisia/química , Artemisia/imunologia , DNA Complementar/genética , DNA Complementar/imunologia , Europa (Continente)/epidemiologia , Galactanos/química , Galactanos/genética , Galactanos/imunologia , Humanos , Imunoglobulina E/imunologia , América do Norte/epidemiologia , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Pólen/química , Estrutura Terciária de Proteína , Rinite Alérgica Sazonal/epidemiologia , Homologia de Sequência de AminoácidosRESUMO
Salmonella contain genes annotated as chitinases; however, their chitinolytic activities have never been verified. We now demonstrate such an activity for a chitinase assigned to glycoside hydrolase family 18 encoded by the SL0018 (chiA) gene in Salmonella enterica Typhimurium SL1344. A C-terminal truncated form of chiA lacking a putative chitin-binding domain was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3) with an N-terminal (His)(6) tag. The purified enzyme hydrolyzes 4-nitrophenyl N,N'-diacetyl-ß-D-chitobioside, 4-nitrophenyl ß-D-N,N',Nâ³-triacetylchitotriose and carboxymethyl chitin Remazol Brilliant Violet but does not act on 4-nitrophenyl N-acetyl-ß-D-glucosaminide, peptidoglycan or 4-nitrophenyl ß-D-cellobioside. Enzyme activity was also characterized by directly monitoring product formation using (1)H-nuclear magnetic resonance which showed that chitin is a substrate with the release of N,N'-diacetylchitobiose. Hydrolysis occurs with the retention of configuration and the enzyme acts on only the ß-anomers of chitooligosaccharide substrates. The enzyme also released N-acetyllactosamine disaccharide from Galß1 â 4GlcNAcß-O-(CH(2))(8)CONH(CH(2))(2)NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.
Assuntos
Amino Açúcares/metabolismo , Quitina/metabolismo , Quitinases/química , Oligossacarídeos/metabolismo , Proteínas Recombinantes/química , Salmonella typhimurium/enzimologia , Quitina/química , Quitinases/genética , Quitinases/metabolismo , Clonagem Molecular , Ensaios Enzimáticos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Oligossacarídeos/química , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Analytical platforms for the fast detection, identification and quantification of circulating drugs with a narrow therapeutic range are vital in clinical pharmacology. As a result of low drug concentrations, analytical tools need to provide high sensitivity and specificity. Dynamic nuclear polarization-NMR (DNP-NMR) in the form of the hyperpolarization-dissolution method should afford the sensitivity and spectral resolution for the direct detection and quantification of numerous isotopically labeled circulating drugs and their metabolites in single liquid-state NMR transients. This study explores the capability of quantitative in vitro DNP-NMR to assay drug metabolites in blood plasma. The lower limit of detection for the anti-epileptic drug (13)C-carbamazepine and its pharmacologically active metabolite (13)C-carbamazepine-10,11-epoxide is 0.08 µg/mL in rabbit blood plasma analyzed by single-scan (13)C DNP-NMR. An internal standard is used for the accurate quantification of drug and metabolite. Comparison of quantitative DNP-NMR data with an established analytical method (liquid chromatography-mass spectrometry) yields a Pearson correlation coefficient r of 0.99. Notably, all DNP-NMR determinations were performed without analyte derivatization or sample purification other than plasma protein precipitation. Quantitative DNP-NMR is an emerging methodology which requires little sample preparation and yields quantitative data with high sensitivity for therapeutic drug monitoring.
Assuntos
Bioensaio/métodos , Carbamazepina/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Plasma/metabolismo , Animais , Calibragem , Carbamazepina/química , Carbamazepina/farmacocinética , Cromatografia Líquida , Interações Medicamentosas , Espectrometria de Massas , Coelhos , Padrões de ReferênciaRESUMO
Upon investigation of Streptococcus pneumoniae serotype 15F capsular polysaccharide (CPS), we discovered that it had a different phosphorylation substituent, namely glycerol-2-phosphate like the other serogroup 15 CPS rather than the originally reported 0.2 equivalent of phosphate or phosphocholine. Furthermore, we also determined the locations of the two previously unassigned O-acetyl groups present in the repeating unit of the 15F CPS, and carried out full NMR assignments of the 15F as well as 15A CPS. Lastly, a biosynthetic analysis of serotypes 15F and 15A was performed and used to make a prediction for the structure of the recently discovered serotype 15D.
Assuntos
Streptococcus pneumoniae , Cápsulas Bacterianas , SorotipagemRESUMO
Capsular polysaccharides (CPS) are the key virulent factors in the pathogenesis of Streptococcus pneumoniae. The previously unknown CPS structures of the pneumococcal serotype 28F and 28A were thoroughly characterized by NMR spectroscopy, chemical analysis and AF4-MALS-dRI. The following repeat unit structures were determined: -4)[α-l-Rhap-[4-P-2-Gro]]-(1-3)-α-d-Sug-[6-P-Cho]-(1-3)-ß-l-Rhap-[2-OAc]-(1-4)-ß-d-Glcp-(1-; 28F: Sug = Glcp, Mw: 540.5 kDa; 28A: Sug = GlcpNAc, Mw: 421.9 kDa; The correlation of CPS structures with biosynthesis showed that glycosyltransferase WciU in serotypes 28F and 28A had different sugar donor specificity toward α-d-Glcp and α-d-GlcNAcp, respectively. Furthermore, latex agglutination tests of de-OAc and de-PO4 CPS were conducted to understand cross-reactions between serogroup 28 with factor antiserum 23d. Interestingly, the de-OAc 28F and 28A CPS can still weakly react with factor antiserum 23d, while de-PO4 CPS did not react with factor antiserum 23d. This indicated that OAc group could affect the affinity and P-2-Gro was crucial for cross-reacting with factor antiserum 23d.
Assuntos
Cápsulas Bacterianas/química , Soros Imunes/imunologia , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/imunologia , Sorogrupo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/genética , Sequência de Aminoácidos , Reações Cruzadas , Glicosiltransferases/química , Testes de Fixação do Látex , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Peso Molecular , Polissacarídeos Bacterianos/biossínteseRESUMO
Lactic acid bacterial exopolysaccharides (EPS) are used in the food industry to improve the stability and rheological properties of fermented dairy products. ß-Lactoglobulin (BLG), the dominant whey protein in bovine milk, is well known to bind small molecules such as fatty acids, vitamins, and flavors, and to interact with neutral and anionic polysaccharides used in food and pharmaceuticals. While sparse data are available on the affinity of EPS-milk protein interactions, structural information on BLG-EPS complexes, including the EPS binding sites, is completely lacking. Here, binding sites on BLG variant A (BLGA), for oligosaccharides prepared by mild acid hydrolysis of two EPS produced by Streptococcus thermophilus LY03 and Lactobacillus delbrueckii ssp. bulgaricus CNRZ 1187, respectively, are identified by NMR spectroscopy and supplemented by isothermal titration calorimetry (ITC) and molecular docking of complexes. Evidence of two binding sites (site 1 and site 2) on the surface of BLGA is achieved for both oligosaccharides (LY03-OS and 1187-OS) through NMR chemical shift perturbations, revealing multivalency of BLGA for EPS. The affinities of LY03-OS and 1187-OS for BLGA gave K D values in the mM range obtained by both NMR (pH 2.65) and ITC (pH 4.0). Molecular docking suggested that the BLGA and EPS complexes depend on hydrogen bonds and hydrophobic interactions. The findings provide insights into how BLGA engages structurally different EPS-derived oligosaccharides, which may facilitate the design of BLG-EPS complexation, of relevance for formulation of dairy products and improve understanding of BLGA coacervation.
RESUMO
Formation of functional nodules is a complex process depending on host-microsymbiont compatibility in all developmental stages. This report uses the contrasting symbiotic phenotypes of Lotus japonicus and L. pedunculatus, inoculated with Mesorhizobium loti or the Bradyrhizobium sp. (Lotus), to investigate the role of Nod factor structure and Nod factor receptors (NFR) for rhizobial recognition, infection thread progression, and bacterial persistence within nodule cells. A key contribution was the use of 800 MHz nuclear magnetic resonance spectroscopy and ultrahigh-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry for Nod factor analysis. The Nod factor decorations at the nonreducing end differ between Bradyrhizobium sp. (Lotus) and M. loti, and the NFR1/NFR5 extracellular regions of L. pedunculatus and L. japonicus were found to vary in amino acid composition. Genetic transformation experiments using chimeric and wild-type receptors showed that both receptor variants recognize the structurally different Nod factors but the later symbiotic phenotype remained unchanged. These results highlight the importance of additional checkpoints during nitrogen-fixing symbiosis and define several amino acids in the LysM domains as expendable for perception of the two differentially carbamoylated Nod factors.
Assuntos
Alphaproteobacteria/fisiologia , Aminoácidos/metabolismo , Bradyrhizobiaceae/fisiologia , Lotus/microbiologia , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Simbiose/fisiologia , Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bradyrhizobiaceae/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Receptores de Lipopolissacarídeos/química , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Lotus/genética , Modelos Moleculares , Dados de Sequência Molecular , Fixação de Nitrogênio/genética , Fenótipo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estrutura Terciária de Proteína , Nódulos Radiculares de Plantas/citologia , Nódulos Radiculares de Plantas/metabolismo , Alinhamento de SequênciaRESUMO
Powerful analytical tools are vital for characterizing the complex molecular changes underlying oncogenesis and cancer treatment. This is particularly true, if information is to be collected in vivo by noninvasive approaches. In the recent past, hyperpolarized (13)C magnetic resonance (MR) spectroscopy has been employed to quickly collect detailed spectral information on the chemical fate of tracer molecules in different tissues at high sensitivity. Here, we report a preclinical study showing that alpha-ketoisocaproic acid (KIC) can be used to assess molecular signatures of tumors with hyperpolarized MR spectroscopy. KIC is metabolized to leucine by the enzyme branched chain amino acid transferase (BCAT), which is found upregulated in some tumors. BCAT is a putative marker for metastasis and a target of the proto-oncogene c-myc. Very different fluxes through the BCAT-catalyzed reaction can be detected for murine lymphoma (EL4) and rat mammary adenocarcinoma (R3230AC) tumors in vivo. EL4 tumors show a more than 7-fold higher hyperpolarized (13)C leucine signal relative to the surrounding healthy tissue. In R3230AC tumor on the other hand branched chain amino acid metabolism is not enhanced relative to surrounding tissues. The distinct molecular signatures of branched chain amino acid metabolism in EL4 and R3230AC tumors correlate well with ex vivo assays of BCAT activity.
Assuntos
Adenocarcinoma/metabolismo , Aminoácidos/metabolismo , Cetoácidos/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Adenocarcinoma/patologia , Animais , Biocatálise , Isótopos de Carbono , Linhagem Celular Tumoral , Feminino , Espectroscopia de Ressonância Magnética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos Lew , Transaminases/metabolismoRESUMO
While the population genetics of inbreeding is fairly well understood, the effects of inbreeding on the physiological and biochemical levels are not. Here we have investigated the effects of inbreeding on the Drosophila melanogaster metabolome. Metabolite fingerprints in males from five outbred and five inbred lines were studied by nuclear magnetic resonance spectroscopy after exposure to benign temperature, heat stress, or cold stress. In both the absence and the presence of temperature stress, metabolite levels were significantly different among inbred and outbred lines. The major effect of inbreeding was increased levels of maltose and decreased levels of 3-hydroxykynurenine and a galactoside [1-O-(4-O-(2-aminoethyl phosphate)-beta-d-galactopyranosyl)-x-glycerol] synthesized exclusively in the paragonial glands of Drosophila species, including D. melanogaster. The metabolomic effect of inbreeding at the benign temperature was related to gene expression data from the same inbred and outbred lines. Both gene expression and metabolite data indicate that fundamental metabolic processes are changed or modified by inbreeding. Apart from affecting mean metabolite levels, inbreeding led to an increased between-line variation in metabolite profiles compared to outbred lines. In contrast to previous observations revealing interactions between inbreeding and environmental stress on gene expression patterns and life-history traits, the effect of inbreeding on the metabolite profile was similar across the different temperature treatments.
Assuntos
Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endogamia , Temperatura , Animais , Galactosídeos/metabolismo , Genética Populacional , Cinurenina/análogos & derivados , Cinurenina/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Maltose/metabolismo , MetabolômicaRESUMO
NMR artifact purging: Modern NMR experiments depend on efficient coherence transfer pathways for their sensitivity and on suppression of undesired pathways leading to artifacts for their spectral clarity. A novel robust adiabatic element suppresses hard-to-get-at artifacts (see picture).
Assuntos
Artefatos , Ressonância Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono/química , Modelos Químicos , Rafinose/química , Processamento de Sinais Assistido por ComputadorRESUMO
ß-lactoglobulin (BLG) is a promiscuous protein in terms of ligand interactions, having several binding sites reported for hydrophobic biomolecules such as fatty acids, lipids, and vitamins as well as detergents. BLG also interacts with neutral and anionic oligo- and polysaccharides for which the binding sites remain to be identified. The multivalency offered by these carbohydrate ligands is expected to facilitate coacervation, an electrostatically driven liquid-liquid phase separation. Using heteronuclear single quantum coherence NMR spectroscopy and monitoring chemical shift perturbations, we observed specific binding sites of modest affinity for alginate oligosaccharides (AOSs) prepared by alginate lyase degradation. Two different AOS binding sites (site 1 and site 2) centered around K75 and K101 were identified for monomeric BLG isoform A (BLGA) at pH 2.65. In contrast, only site 1 around K75 was observed for dimeric BLGA at pH 4.0. The data suggest a pH-dependent mechanism whereby both the BLGA dimer-monomer equilibrium and electrostatic interactions are exploited. This variability allows for control of coacervation and particle formation of BLGA/alginate mixtures via directed polysaccharide bridging of AOS binding sites and has implication for molecular network formation. The results are valuable for design of polyelectrolyte-based BLG particles and coacervates for carrying nutraceuticals and modulating viscosity in dairy products by use of alginates.