Lidia Nieto

Lignin Composition and Structure in Young versus Adult Eucalyptus globulus Plants

Lignin changes during plant growth were investigated in a selected Eucalyptus globulus clone. The lignin composition and structure were studied in situ by a new procedure enabling the acquisition of two-dimensional nuclear magnetic resonance (2D-NMR) spectra on wood gels formed in the NMR tube as well as by analytical pyrolysis-gas chromatography-mass spectrometry. In addition, milled-wood lignins were isolated and analyzed by 2D-NMR, pyrolysis-gas chromatography-mass spectrometry, and thioacidolysis. The data indicated that p-hydroxyphenyl and guaiacyl units are deposited at the earlier stages, whereas the woods are enriched in syringyl (S) lignin during late lignification. Wood 2D-NMR showed that β-O-4′ and resinol linkages were predominant in the eucalypt lignin, whereas other substructures were present in much lower amounts. Interestingly, open β-1′ structures could be detected in the isolated lignins. Phenylcoumarans and cinnamyl end groups were depleted with age, spirodienone abundance increased, and the main substructures (β-O-4′ and resinols) were scarcely modified. Thioacidolysis revealed a higher predominance of S units in the ether-linked lignin than in the total lignin and, in agreement with NMR, also indicated that resinols are the most important nonether linkages. Dimer analysis showed that most of the resinol-type structures comprised two S units (syringaresinol), the crossed guaiacyl-S resinol appearing as a minor substructure and pinoresinol being totally absent. Changes in hemicelluloses were also shown by the 2D-NMR spectra of the wood gels without polysaccharide isolation. These include decreases of methyl galacturonosyl, arabinosyl, and galactosyl (anomeric) signals, assigned to pectin and related neutral polysaccharides, and increases of xylosyl (which are approximately 50% acetylated) and 4-O-methylglucuronosyl signals.

Solution structures of chemoenzymatically synthesized heparin and its precursors

We report the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly labeled [(13)C,(15)N]N-acetylheparosan (-GlcA(1,4)GlcNAc-) prepared from E. coli K5. Glycosaminoglycan (GAG) precursors and heparin were formed from N-acetylheparosan by the following steps: chemical N-deacetylation and N-sulfonation leading to N-sulfoheparosan (-GlcA(1,4)GlcNS-); enzyme-catalyzed C5-epimerization and 2-O-sulfonation leading to undersulfated heparin (-IdoA2S(1,4)GlcNS-); enzymatic 6-O-sulfonation leading to the heparin backbone (-IdoA2S(1,4)GlcNS6S-); and selective enzymatic 3-O-sulfonation leading to the anticoagulant heparin, containing the GlcNS6S3S residue. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution structure of [(13)C,(15)N]N-acetylheparosan, precursors, and heparin. Isotopic enrichment was found to provide well-resolved (13)C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labeled heparin was indistinguishable from heparin derived from animal tissues and is a novel reagent for studying the interaction of heparin with proteins.

HSQC-NMR analysis of lignin in woody (Eucalyptus globulus and Picea abies) and non-woody (Agave sisalana) ball-milled plant materials at the gel state

Abstract In situ analysis of lignin by 2D NMR of whole plant material was carried out by swelling finely ball-milled samples in deuterated dimethylsulfoxide (DMSO-d6 ) and sonicated so that a gel was formed in the NMR analysis tube. Solution HSQC NMR spectra of different plant materials representative for hardwood (Eucalyptus globulus), softwood (Picea abies), and non-woody plants (Agave sisalana) are presented here. The spectra show signals corresponding to those of the main plant constituents, such as lignin and polysaccharides. The lignin signals were assigned by comparing the HSQC spectra of the whole plant materials with the HSQC spectra of their respective milled-wood lignins (MWLs). In general terms, the major lignin structural features, such as the relative abundances of the main lignin substructures, the syringyl/guaiacyl ratios and the extent of γ-acetylation of the lignin side-chain observed in the HSQC spectra of the whole plant materials, matched those obtained from the HSQC spectra of the isolated MWLs. Therefore, this technique, which needs only minor amounts of lignocellulosic material and minimal sample preparation, can be useful for the rapid screening of plant lignins without the need for tedious and time-consuming lignin isolation procedures.

HSQC-NMR analysis of lignin in woody (Eucalyptus globulus and Picea abies) and non-woody (Agave sisalana) ball-milled plant materials at the gel state 10th EWLP, Stockholm, Sweden, August 25–28, 2008

Abstract In situ analysis of lignin by 2D NMR of whole plant material was carried out by swelling finely ball-milled samples in deuterated dimethylsulfoxide (DMSO-d6 ) and sonicated so that a gel was formed in the NMR analysis tube. Solution HSQC NMR spectra of different plant materials representative for hardwood (Eucalyptus globulus), softwood (Picea abies), and non-woody plants (Agave sisalana) are presented here. The spectra show signals corresponding to those of the main plant constituents, such as lignin and polysaccharides. The lignin signals were assigned by comparing the HSQC spectra of the whole plant materials with the HSQC spectra of their respective milled-wood lignins (MWLs). In general terms, the major lignin structural features, such as the relative abundances of the main lignin substructures, the syringyl/guaiacyl ratios and the extent of γ-acetylation of the lignin side-chain observed in the HSQC spectra of the whole plant materials, matched those obtained from the HSQC spectra of the isolated MWLs. Therefore, this technique, which needs only minor amounts of lignocellulosic material and minimal sample preparation, can be useful for the rapid screening of plant lignins without the need for tedious and time-consuming lignin isolation procedures.

Polymerization of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility

The ability of laccases from Trametes villosa (TvL), Myceliophthora thermophila (MtL), Trametes hirsuta (ThL) and Bacillus subtilis (BsL) to improve the dispersion properties of calcium lignosulfonates 398 in the presence of HBT as a mediator was investigated. Size exclusion chromatography showed an extensive increase in molecular weight of the samples incubated with TvL and ThL by 107% and 572% from 28400 Da after 17h of incubation, respectively. Interestingly, FTIR spectroscopy, (13)C NMR and Py-GC/MS analysis of the treated samples suggested no substantial changes in the aromatic signal of the lignosulfonates, a good indication of the ability of TvL/ThL-HBT systems to limit their effect on functional groups without degrading the lignin backbone. Further, the enzymatic treatments led to a general increase in the dispersion properties, indeed a welcome development for its application in polymer blends.

Molecular Basis for Inhibition of GH84 Glycoside Hydrolases by Substituted Azepanes: Conformational Flexibility Enables Probing of Substrate Distortion

Here we report the synthesis of a series of polyhydroxylated 3- and 5-acetamido azepanes and detail the molecular basis of their inhibition of family 84 glycoside hydrolases. These family 84 enzymes include human O-GlcNAcase, an enzyme involved in post-translational processing of intracellular proteins modified by O-linked beta-N-acetylglucosamine residues. Detailed structural analysis of the binding of these azepanes to BtGH84, a bacterial homologue of O-GlcNAcase, highlights their conformational flexibility. Molecular mechanics and molecular dynamics calculations reveal that binding to the enzyme involves significant conformational distortion of these inhibitors from their preferred solution conformations. The binding of these azepanes provides structural insight into substrate distortion that likely occurs along the reaction coordinate followed by O-GlcNAcase during glycoside hydrolysis. This class of inhibitors may prove to be useful probes for evaluating the conformational itineraries of glycosidases and aid the development of more potent and specific glycosidase inhibitors.

Insights into the interaction of discodermolide and docetaxel with tubulin. Mapping the binding sites of microtubule-stabilizing agents by using an integrated NMR and computational approach.

The binding interactions of two antitumor agents that target the paclitaxel site, docetaxel and discodermolide, to unassembled α/β-tubulin heterodimers and microtubules have been studied using biochemical and NMR techniques. The use of discodermolide as a water-soluble paclitaxel biomimetic and extensive NMR experiments allowed the detection of binding of microtubule-stabilizing agents to unassembled tubulin α/β-heterodimers. The bioactive 3D structures of docetaxel and discodermolide bound to α/β-heterodimers were elucidated and compared to those bound to microtubules, where subtle changes in the conformations of docetaxel in its different bound states were evident. Moreover, the combination of experimental TR-NOE and STD NMR data with CORCEMA-ST calculations indicate that docetaxel and discodermolide target an additional binding site at the pore of the microtubules, which is different from the internal binding site at the lumen previously determined by electron crystallography. Binding to this pore site can then be considered as the first ligand-protein recognition event that takes place in advance of the drug internalization process and interaction with the lumen of the microtubules.

Heparin modulates the mitogenic activity of fibroblast growth factor by inducing dimerization of its receptor. a 3D view by using NMR.

In vitro mitogenesis assays have shown that sulfated glycosaminoglycans (GAGs; heparin and heparan sulfate) cause an enhancement of the mitogenic activity of fibroblast growth factors (FGFs). Herein, we report that the simultaneous presence of FGF and the GAG is not an essential requisite for this event to take place. Indeed, preincubation with heparin (just before FGF addition) of cells lacking heparan sulfate produced an enhancing effect equivalent to that observed when the GAG and the protein are simultaneously added. A first structural characterization of this effect by analytical ultracentrifugation of a soluble preparation of the heparin‐binding domain of fibroblast growth factor receptor 2 (FGFR2) and a low molecular weight (3 kDa) heparin showed that the GAG induces dimerization of FGFR2. To derive a high resolution structural picture of this molecular recognition process, the interactions of a soluble heparin‐binding domain of FGFR2 with two different homogeneous, synthetic, and mitogenically active sulfated GAGs were analyzed by NMR spectroscopy. These studies, assisted by docking protocols and molecular dynamics simulations, have demonstrated that the interactions of these GAGs with the soluble heparin‐binding domain of FGFR induces formation of an FGFR dimer; its architecture is equivalent to that in one of the two distinct crystallographic structures of FGFR in complex with both heparin and FGF1. This preformation of the FGFR dimer (with similar topology to that of the signaling complex) should favor incorporation of the FGF component to form the final assemblage of the signaling complex, without major entropy penalty. This cascade of events is probably at the heart of the observed activating effect of heparin in FGF‐driven mitogenesis.

Structure of micelle-bound adrenomedullin: A first step toward the analysis of its interactions with receptors and small molecules†‡

Adrenomedullin (AM) is a regulatory peptide which plays many physiological roles including vasodilatation, bronchodilatation, hormone secretion regulation, growth, apoptosis, angiogenesis, and antimicrobial activities, among others. These regulatory activities make AM a relevant player in the pathophysiology of important diseases such as cardiovascular and renal conditions, cancer, and diabetes. Therefore, molecules that target the AM system have been proposed as having therapeutic potential. To guide the design and characterization of such molecules, we elucidated the three-dimensional structure of AM in a membrane mimicking medium using NMR spectroscopy methods. Under the employed experimental conditions, the structure can be described as composed by a central α-helical region, spanning about one third of its total length, flanked by two disordered segments at both N- and C-termini. The structure of AM in water is completely disordered. The 22-34 region of AM has a general tendency to adopt a helical structure under the employed experimental conditions. Furthermore, the study of the interaction of AM with two of its modulators has also been performed by using chemical shift perturbation analysis NMR methods with two-dimensional (2D)-TOCSY experiments, assisted with molecular modeling protocols. We expect these results will help in better understanding the interactions of AM with its receptor and binding proteins/molecules and in the development of novel modulators of AM activities.

Conformational Selection of the AGA*IAM Heparin Pentasaccharide when Bound to the Fibroblast Growth Factor Receptor

The interaction of the synthetic pentasaccharide AGA*IA(M) (GlcNS,6S-GlcA-GlcNS,3S,6S-IdoA2S-GlcNS,6S-Me) with the extracellular Ig2 domain of the fibroblast growth factor receptor (FGFR2) has been studied by NMR and computational methods. Analysis of the heparin pentasaccharide in the free state and in the complex indicates the existence of a conformational selection process. Although an equilibrium exists between the (1)C(4) and (2)S(0) conformers (ratio 60:40) of the 2-O-sulfo-α-L-iduronate ring (IdoA2S) in the free state, FGFR2 selects only the unique twisted-boat (2)S(0) conformation of this IdoA2S residue. In addition, the protein residues involved in the binding with AGA*IA(M) have also been characterized. The NMR results obtained, from both the ligand and protein perspective, were employed to model the bound conformation of the pentasaccharide by a combined docking and molecular dynamic simulation approach.