POLYS 2.0: An open source software package for building three-dimensional structures of polysaccharides.

This article describes an update of POLYS, the POLYSaccharide builder, for generating three-dimensional structures of polysaccharides and complex carbohydrates (Engelsen et al., Biopolymers 1996, 39, 417-433). POLYS is written in portable ANSI C and is now released under an open source license. Using this software, complex branched carbohydrate structures and polysaccharides can be constructed from their primary structure and the relevant monosaccharides stored in database containing information on optimized glycosidic linkage geometries. The constructed three-dimensional structures are described as Cartesian coordinate files which can be used as input to other molecular modeling software. The new version of POLYS includes a large database of monosaccharides and a helical generator to build and optimize regular single helix or double helix structures. To demonstrate the efficiency of POLYS to build carbohydrate structures, four examples of increasing complexity are presented in the manuscript, from simple alpha glucans over complex starch fragments and the double helical structure of amylopectin to the mega-oligosaccharide RhamnoGalacturonan II.

Computational simulations of the Trichoderma reesei cellobiohydrolase I acting on microcrystalline cellulose Ibeta: the enzyme-substrate complex.

Cellobiohydrolases are the dominant components of the commercially relevant Trichoderma reesei cellulase system. Although natural cellulases can totally hydrolyze crystalline cellulose to soluble sugars, the current enzyme loadings and long digestion times required render these enzymes less than cost effective for biomass conversion processes. It is clear that cellobiohydrolases must be improved via protein engineering to reduce processing costs. To better understand cellobiohydrolase function, new simulations have been conducted using charmm of cellobiohydrolase I (CBH I) from T.reesei interacting with a model segment (cellodextrin) of a cellulose microfibril in which one chain from the substrate has been placed into the active site tunnel mimicking the hypothesized configuration prior to final substrate docking (i.e., the +1 and +2 sites are unoccupied), which is also the structure following a catalytic bond scission. No tendency was found for the protein to dissociate from or translate along the substrate surface during this initial simulation, nor to align with the direction of the cellulose chains. However, a tendency for the decrystallized cellodextrin to partially re-anneal into the cellulose surface hints that the arbitrary starting configuration selected was not ideal.

Starch phosphorylation--maltosidic restrains upon 3'- and 6'-phosphorylation investigated by chemical synthesis, molecular dynamics and NMR spectroscopy.

Phosphorylation is the only known in vivo substitution of starch, yet no structural evidence has been provided to explain its implications of the amylosidic backbone and its stimulating effects on starch degradation in plants. In this study, we provide evidence for a major influence on the glucosidic bond in starch specifically induced by the 3-O-phosphate. Two phosphorylated maltose model compounds were synthesized and subjected to combined molecular dynamics (MD) studies and 950 MHz NMR studies. The two phosphorylated disaccharides represent the two possible phosphorylation sites observed in natural starches, namely maltose phosphorylated at the 3'- and 6'-position (maltose-3'-O-phosphate and maltose-6'-O-phosphate). When compared with maltose, both of the maltose-phosphates exhibit a restricted conformational space of the alpha(1-->4) glycosidic linkage. When maltose is phosphorylated in the 3'-position, MD and NMR show that the glucosidic space is seriously restricted to one narrow potential energy well which is strongly offset from the global potential energy well of maltose and almost 50 degrees degrees from the Phi angle of the alpha-maltose crystal structure. The driving force is primarily steric, but the configuration of the structural waters is also significantly altered. Both the favored conformation of the maltose-3'-phosphate and the maltose-6'-phosphate align well into the 6-fold double helical structure of amylopectin when the effects on the glucosidic bond are not taken into account. However, the restrained geometry of the glucosidic linkage of maltose-3'-phosphate cannot be accommodated in the helical structure, suggesting a major local disturbing effect, if present in the starch granule semi-crystalline lattice.

Structure and hydration of the amylopectin trisaccharide building blocks--Synthesis, NMR, and molecular dynamics.

To gain insight into the molecular details and hydration of amylopectin, the five constituting trisaccharides have been chemically synthesized as their methyl alpha-glycosides. All five trisaccharides were subjected to 950 MHz NMR spectroscopy for complete assignment and nanosecond molecular dynamics trajectories were calculated to study the structure and dynamics of the trisaccharides in aqueous solution. Systematic analysis of the simulation data revealed several examples of bridging water molecules playing an important role in the stabilization of specific amylopectin conformations, which was also supported by the experimental NMR data such as interresidue NOE's and heteronuclear scalar couplings between nuclei from neighboring residues. Although alpha-maltotriose, alpha-iso-maltotriose, alpha-panose and alpha-isopanose are relatively well characterized structures, the study also includes one less characterized trisaccharide with the structure alphaGlcp(1-->4)alphaGlcp(1-->6)alphaGlcp. This trisaccharide, tentatively labelled alpha-forkose, is located at the branch point of amylopectin, forking the amylopectin into two strands that align into double-helical segments. The results show that the conformation of alpha-forkose takes a natural bend form which fits well into the structure of the double-helical segment of amylopectin. As the only trisaccharide in this study the structure of alpha-forkose is not significantly influenced by the hydration. In contrast, alpha-isopanose takes a restricted, but rather extended form due to an exceptionally strong localized water density. The two homo-linkage oligomers, alpha-maltotriose and alpha-iso-maltotriose, showed to be the most extended and the most flexible trimers, respectively, providing regular structure for crystalline domains and maximum linker flexibility for amorphous domains.