Conformational properties of l-fucose and the tetrasaccharide building block of the sulfated l-fucan from Lytechinus variegatus.

Although the 3D structure of carbohydrates is known to contribute to their biological roles, conformational studies of sugars are challenging because their chains are flexible in solution and consequently the number of 3D structural restraints is limited. Here, we investigate the conformational properties of the tetrasaccharide building block of the Lytechinus variegatus sulfated fucan composed of the following structure [l-Fucp4(SO3-)-α(1-3)-l-Fucp2,4(SO3-)-α(1-3)-l-Fucp2(SO3-)-α(1-3)-l-Fucp2(SO3-)] and the composing monosaccharide unit Fucp, primarily by nuclear magnetic resonance (NMR) experiments performed at very low temperatures and using H2O as the solvent for the sugars rather than using the conventional deuterium oxide. By slowing down the fast chemical exchange rates and forcing the protonation of labile sites, we increased the number of through-space 1H-1H distances that could be measured by NMR spectroscopy. Following this strategy, additional conformational details of the tetrasaccharide and l-Fucp in solution were obtained. Computational molecular dynamics was performed to complement and validate the NMR-based measurements. A model of the NMR-restrained 3D structure is offered for the tetrasaccharide.

Crystal structure and thermodynamic dissection of chitin oligosaccharide binding to the LysM module of chitinase-A from Pteris ryukyuensis.

We determined the crystal structure of a LysM module from Pteris ryukyuensis chitinase-A (PrLysM2) at a resolution of 1.8 Å. Structural and binding analysis of PrLysM2 indicated that this module recognizes chitin oligosaccharides in a shallow groove comprised of five sugar-binding subsites on one side of the molecule. The free energy changes (ΔGr°) for binding of (GlcNAc)6, (GlcNAc)5, and (GlcNAc)4 to PrLysM2 were determined to be -5.4, -5,4 and -4.6 kcal mol-1, respectively, by ITC. Thermodynamic dissection of the binding energetics of (GlcNAc)6 revealed that the driving force is the enthalpy change (ΔHr° = -11.7 ± 0.2 kcal/mol) and the solvation entropy change (-TΔSsolv° = -5.9 ± 0.6 kcal/mol). This is the first description of thermodynamic signatures of a chitin oligosaccharide binding to a LysM module.

Produccion de beta-glucosidasas por cultivos de bacterias termofilas indigenas del altiplano boliviano / beta-Glucoside production by thermophilic bacteria indigenous the bolivian altiplano culture

Las beta-glucosidasas son enzimas que poseen actividad hidrolitica y transferasa o transglucosidasa. Tienen diversas aplicaciones; en la biosintesis de oligosacaridos, produccion de etanol utilizando residuos agricolas y en la industria de vinos. La aplicacion industrial, sin embargo, requiere estabilidad a temperaturas elevadas, por lo que los microorganismos termofilos tienen gran interes. El proposito de esta investigacion es el de optimizar el medio de cultivo anaerobio de bacterias termofilas, para aumentar la produccion de beta-glucosidasas. Esta enzima es producida por tres aislados bacterianos: FT3, 2B y P5 los cuales fueron aislados de la region andina de Bolivia. El aislado bacteriano FT3 mostro una actividad beta-glucosidasa de 0,35 [UI/mL]. Se tomaron como variables dentro de la optimizacion del medio de cultivo las fuentes de nitrogeno y de carbono, y el pH. Asi tambien se probaron dos sistemas de cultivo: celulas libres y encapsuladas. Empleando extracto de levadura como fuente de nitrogeno se obtuvo una actividad de 0,52 [UI/mL]. En la optimizacion del pH del medio de cultivo se obtuvo una actividad de 0,81 [UI/mL] a pH 5. Como fuente de carbono se eligieron los hidrolizados de paja de trigo y paja de quinoa lleg¨¢ndose a obtener actividades de 1,27 y 1,34 [UI/mL] respectivamente. Se establecio que la localizacion celular de la enzima beta-glucosidasa es extracelular y presenta estabilidad hasta una temperatura de 80 ºC y un pH de 7.

The beta-glucosidases possess hydrolytic and transferase activity or transglucosidase. They have various applications; such as biosynthesis of oligosaccharides, production of ethanol using agricultural residues and wine industry. However for industrial application, stability to high temperatures is needed. Therefore a great interesting in the thermophile microorganism study exist. The purpose of this research is to optimize the culture medium of thermophilic anaerobic bacteria to increase the production of beta-glucosidase. This enzyme is produced by three isolate bacterial FT3, 2B and P5 which were isolated from the Andean region of Bolivia. FT3 isolate showed beta-glucosidase activity of 0.35 [IU/mL]. In regards to the optimization of culture medium variables such as nitrogen source, carbon source and pH were taken into account and also the combination with free and encapsulated bacterial cells. Yeast extract was the selected source of nitrogen obtaining an activity of 0.52 [IU/ mL]. The optimal pH was 5 obtaining an activity of 0.81 [IU/mL]. The selected carbon source was the hydrolyzed wheat straw and quinoa straw obtaining activities of 1.27 and 1.34 [IU/mL], respectively. The cellular localization of beta-glucosidase enzyme is extracellular and provides stability to temperature of 80 ºC and stability at pH 7.

Starch with a slow digestion property produced by altering its chain length, branch density, and crystalline structure.

The hypothesis of increasing the branch density of starch to reduce its digestion rate through partial shortening of amylopectin exterior chains and the length of amylose was investigated. Starch products prepared using beta-amylase, beta-amylase and transglucosidase, maltogenic alpha-amylase, and maltogenic alpha-amylase and transglucosidase showed significant reduction of rapidly digested starch by 14.5%, 29.0%, 19.8%, and 31.0% with a concomitant increase of slowly digested starch by 9.0%, 19.7%, 5.7%, and 11.0%, respectively. The resistant starch content increased from 5.1% to 13.5% in treated starches. The total contents of the prebiotics isomaltose, isomaltotriose, and panose (Isomaltooligosaccharides) were 2.3% and 5.5%, respectively, for beta-amylase/transglucosidase- and maltogenic alpha-amylase/transglucosidase-treated starches. The molecular weight distribution of enzyme-treated starches and their debranched chain length distributions, analyzed using high-performance size-exclusion chromatography with multiangle laser light scattering and refractive index detection (HPSEC-MALLS-RI) and HPSEC-RI, showed distinctly different patterns among starches with different enzyme treatments. A larger proportion of low molecular weight fractions appeared in starches treated additionally with transglucosidase. All enzyme-treated starches showed a mixture of B- and V-type X-ray diffraction patterns, and 1H NMR spectra showed a significant increase of alpha-1,6 linkages. Both the increase of the starch branch density and the crystalline structure in the treated starches likely contribute to their slow digestion property.

Crystal structures of a mutant maltotetraose-forming exo-amylase cocrystallized with maltopentaose.

The three-dimensional structures of the catalytic residue Glu219-->Gln mutant of Pseudomonas stutzeri maltotetraose-forming exo-alpha-amylase, and its complex with carbohydrate obtained by cocrystallization with maltopentaose were determined. Two crystal forms were obtained for the complexed enzyme, and a bound maltotetraose was found in each. The structures were analyzed at 2.2 A and 1.9 A resolution, respectively for the uncomplexed and complexed mutant. These structures were compared with the wild-type enzyme structure. In the complexed crystals, the maltotetraose was firmly bound, extensively interacting with the amino acid environments in the active cleft. The non-reducing end glucose unit was hydrogen bonded to the side-chain of Asp160 and the main-chain nitrogen of Gly158, which seem to be predominantly required for the recognition of the non-reducing end of the substrate that determines the exo-wise degradation of this enzyme. The reducing end glucose unit of bound maltotetraose showed clear deformation, adopting a half-chair conformation with extensive hydrogen bonds to surrounding polypeptides. The C1-atom of this deformed glucose unit lies very close to Asp193OD1 with a distance of 2.6 A. The catalytic residue Asp294 is firmly hydrogen-bonded to the O2 and O3-hydroxyl groups of the deformed reducing end glucose unit. Upon binding of the carbohydrate, small but significant induced fits were observed in the regions of Asp294, Phe156, Ile157, and Asp160. Possible roles of the three catalytic residues are also discussed.

Structure of the major oligosaccharide from the lipooligosaccharide of Haemophilus ducreyi strain 35000 and evidence for additional glycoforms.

Haemophilus ducreyi is a sexually transmitted pathogen that colonizes the genital epithelium in humans, causing genital ulcers or chancroid. Its surface lipooligosaccharides (LOSs) have been shown to play a role in ulcer formation and may also be important in cell adhesion and invasion of host tissue. Earlier we presented a preliminary structure of the major LOS from strain 35000 that suggested the presence of terminal lactosamine [Melaugh, W., Phillips, N.J., Campagnari, A.A., Karalus, R., & Gibson, B. W. (1992) J. Biol. Chem. 267, 13434-13439]. We have now confirmed this structure and assigned the anomeric linkages by 2D NMR studies. In addition to this major structure, analysis by electrospray ionization mass spectrometry of both O-deacylated LOSs and the oligosaccharides released after treatment with mild acid indicates the presence of several other LOS glycoforms. These glycoforms constitute a series of both truncated and elongated analogs of the major oligosaccharide determined by NMR. One of these glycoforms exists as a smaller oligosaccharide corresponding to the major structure minus terminal galactose. Three other glycoforms appear as larger molecular weight species formed by the addition of phosphoethanolamine, N-acetylhexosamine, and N-acetylhexosamine plus hexose. Two sialylated glycoforms were also identified and subsequently confirmed by treatment with neuraminidase, but these glycoforms were not found in the released oligosaccharide pool due to the acid lability of of sialic acid. This study clearly indicates that the LOSs from H. ducreyi strain 35000 exist as a heterogeneous population whose structures differ primarily in their phosphorylation states and terminal sugars and whose terminal glycan structures can resemble those of human antigens.

Calculated conformations of sialyl-Le(x)- and sialyl-Le(a)-lactones.

The minimum energy conformations of the four sterically reasonable SLe(x) and SLe(a) lactones were calculated using the molecular mechanics force-field MM2(91). The tetrasaccharide lactone involving the 3- and 2-position of the Gal moiety was found to be more stable than the 3,4-lactone both for SLe(x) and SLe(a).