Enantioselective Binding of Propranolol and Analogues Thereof to Cellobiohydrolase Cel7A.

At the catalytic site for the hydrolysis of cellulose the enzyme cellobiohydrolase Cel7A binds the enantiomers of the adrenergic beta-blocker propranolol with different selectivity. Methyl-to-hydroxymethyl group modifications of propranolol, which result in higher affinity and improved selectivity, were herein studied by 1 H,1 H and 1 H,13 C scalar spin-spin coupling constants as well as utilizing the nuclear Overhauser effect (NOE) in conjunction with molecular dynamics simulations of the ligands per se, which showed the presence of all-antiperiplanar conformations, except for the one containing a vicinal oxygen-oxygen arrangement governed by the gauche effect. For the ligand-protein complexes investigated by NMR spectroscopy using, inter alia, transferred NOESY and saturation-transfer difference (STD) NMR experiments the S-isomers were shown to bind with a higher affinity and a conformation similar to that preferred in solution, in contrast to the R-isomer. The fact that the S-form of the propranolol enantiomer is pre-arranged for binding to the protein is also observed for a crystal structure of dihydroxy-(S)-propranolol and Cel7A presented herein. Whereas the binding of propranolol is entropy driven, the complexation with the dihydroxy analogue is anticipated to be favored also by an enthalpic term, such as for its enantiomer, that is, dihydroxy-(R)-propranolol, because hydrogen-bond donation replaces the corresponding bonding from hydroxyl groups in glucosyl residues of the natural substrate. In addition to a favorable entropy component, albeit lesser in magnitude, this represents an effect of enthalpy-to-entropy compensation in ligand-protein interactions.

Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development.

Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture.

Selection and phenotypic characterization of a core collection of Brachypodium distachyon inbred lines.

BACKGROUND: The model grass Brachypodium distachyon is increasingly used to study various aspects of grass biology. A large and genotypically diverse collection of B. distachyon germplasm has been assembled by the research community. The natural variation in this collection can serve as a powerful experimental tool for many areas of inquiry, including investigating biomass traits. RESULTS: We surveyed the phenotypic diversity in a large collection of inbred lines and then selected a core collection of lines for more detailed analysis with an emphasis on traits relevant to the use of grasses as biofuel and grain crops. Phenotypic characters examined included plant height, growth habit, stem density, flowering time, and seed weight. We also surveyed differences in cell wall composition using near infrared spectroscopy (NIR) and comprehensive microarray polymer profiling (CoMPP). In all cases, we observed extensive natural variation including a two-fold variation in stem density, four-fold variation in ferulic acid bound to hemicellulose, and 1.7-fold variation in seed mass. CONCLUSION: These characterizations can provide the criteria for selecting diverse lines for future investigations of the genetic basis of the observed phenotypic variation.

The importance of pyroglutamate in cellulase Cel7A.

The commercialization of lignocellulosic biofuels relies in part on the ability to engineer cellulase enzymes to have properties compatible with practical processing conditions. The cellulase Cel7A has been a common engineering target because it is present in very high concentrations in commercial cellulase cocktails. Significant effort has thus been focused on its recombinant expression. In particular, the yeast Saccharomyces cerevisiae has often been used both in the engineering and basic study of Cel7A. However, the expression titer and extent of glycosylation of Cel7A expressed in S. cerevisiae vary widely for Cel7A genes from different organisms, and the recombinant enzymes tend to be less active and less stable than their native counterparts. These observations motivate further study of recombinant expression of Cel7A in S. cerevisiae. Here, we compare the properties of Cel7A from Talaromyces emersonii expressed in both the budding yeast S. cerevisiae and the filamentous fungus Neurospora crassa. The Cel7A expressed in N. crassa had a higher melting temperature (by 10°C) and higher specific activity (twofold at 65°C) than the Cel7A expressed in S. cerevisiae. We examined several post-translational modifications and found that the underlying cause of this disparity was the lack of N-terminal glutamine cyclization in the Cel7A expressed in S. cerevisiae. Treating the enzyme in vitro with glutaminyl cyclase improved the properties of Cel7A expressed in S. cerevisiae to match those of Cel7A expressed in N. crassa.

Overexpression of the grapevine PGIP1 in tobacco results in compositional changes in the leaf arabinoxyloglucan network in the absence of fungal infection.

BACKGROUND: Constitutive expression of Vitis vinifera polygalacturonase-inhibiting protein 1 (Vvpgip1) has been shown to protect tobacco plants against Botrytis cinerea. Evidence points to additional roles for VvPGIP1, beyond the classical endopolygalacturonase (ePG) inhibition mechanism, in providing protection against fungal infection. Gene expression and biochemical datasets previously obtained, in the absence of infection, point to the cell wall, and particularly the xyloglucan component of transgenic VvPGIP1 lines as playing a role in fungal resistance. RESULTS: To elucidate the role of wall-associated processes in PGIP-derived resistance pre-infection, a wall profiling analysis, using high-throughput and fractionation techniques, was performed on healthy leaves from wild-type and previously characterized transgenic lines. The cell wall structure profile during development was found to be altered in the transgenic lines assessed versus the wild-type plants. Immunoprofiling revealed subtle changes in pectin and cellulose components and marked changes in the hemicellulose matrix, which showed reduced binding in transgenic leaves of VvPGIP1 expressing plants. Using an enzymatic xyloglucan oligosaccharide fingerprinting technique optimized for tobacco arabinoxyloglucans, we showed that polysaccharides of the XEG-soluble domain were modified in relative abundance for certain oligosaccharide components, although no differences in ion profiles were evident between wild-type and transgenic plants. These changes did not significantly influence plant morphology or normal growth processes compared to wild-type lines. CONCLUSIONS: VvPGIP1 overexpression therefore results in cell wall remodeling and reorganization of the cellulose-xyloglucan network in tobacco in advance of potential infection.

Thermodynamical studies of designed ligands binding to Cel7A using partial-filling capillary electrophoresis.

A convenient experimental method for thermodynamical studies based on partial-filling affinity CE is presented. The advantages of this approach are the possibility to determine binding energies from relatively weak interactions as well as the small amounts of samples consumed. In order to explore the affinity and selectivity of the cellobiohydrolase Cel7A, a number of propranolol analogues were recently designed. The affinities of a selection of these ligands were determined in the temperature interval 15-40 degrees C, and DeltaG degrees , DeltaH degrees and DeltaS degrees were obtained by means of Van't Hoff plots. Through these experiments, the importance of the entropy contribution in the complexation between the ligands and Cel7A has been demonstrated.

pH dependency of ligand binding to cellobiohydrolase 1 (Cel7A). Affinity, selectivity and inhibition for designed propranolol analogues.

The affinity and enantioselectivity have been determined for designed propranolol derivatives as ligands for Cel7A by capillary electrophoresis (CE) at pH 7.0. These results have been compared to measurements at pH 5.0. In agreement with previous studies, the affinity increased at the higher pH. However, the affinity was not as dependent of the ligand structure at pH 7.0 as at pH 5.0, and the selectivity was generally decreased. Instead, at pH 7.0, the changes in binding were mainly dependent on the presence of additional dihydroxyl groups, indicating an increased importance of the electrostatic interactions. To evaluate the pH dependent variations in binding, changes in both the ligand and in the enzyme had to be taken into account. To ensure that the ligands had the same charge in all measurements, pKa-values of all compounds were determined. The ligand-protein interaction has also been studied by inhibition experiments at both pHs to evaluate the specific binding to the active site when competing with the substrate p-nitrophenyl lactoside (pNPL). With support of docking computations we propose a hypothesis on the effect of the ligand structure and pH dependency of the binding and selectivity of amino alcohols to Cel7A.

New propranolol analogues: binding and chiral discrimination by cellobiohydrolase Cel7A.

Novel propranolol analogues have been designed and synthesised and their enantioselective binding to the cellulose degrading enzyme, Cel7A, has been evaluated. Affinity and enantioselectivity have been determined by capillary electrophoresis experiments. Ligands with significantly improved affinity and selectivity have been obtained and an analysis of the results has led to insights concerning the relation between the changes in ligand structure and selectivity as well as affinity to the protein.