Oligo- and poly-saccharides have a large number of important biological functions, and they occur in natural composite materials, such as plant cell walls, where they self-assemble during ...biosynthesis in a poorly understood manner. They can also be used for the formation of artificial composite materials with industrial applications. Fundamental and applied research in biology and nanobiotechnology would benefit from the possibility of synthesizing tailor-made oligo-/poly-saccharides. In the present paper, we demonstrate that such syntheses are possible using genetically modified glycoside hydrolases, i.e. glycosynthases. The ability of the endoglycosynthase derived from Bacillus (1-->3,1-->4)-beta-D-glucanase to catalyse self-condensation of sugar donors was exploited for the in vitro synthesis of a regular polysaccharide. The specificity of the enzyme allowed the polymerization of alpha-laminaribiosyl fluoride via the formation of (1-->4)-beta-linkages to yield a new linear crystalline (1-->3,1-->4)-beta-D-glucan with a repeating 4betaG3betaG unit. MS and methylation analyses indicated that the in vitro product consisted of a mixture of oligosaccharides, the one having a degree of polymerization of 12 being the most abundant. Morphological characterization revealed that the (1-->3,1-->4)-beta-D-glucan forms spherulites which are composed of platelet crystals. X-ray and electron diffraction analyses allowed the proposition of a putative crystallographic structure which corresponds to a monoclinic unit cell with a =0.834 nm, b =0.825 nm, c =2.04 nm and gamma=90.5 degrees. The dimensions of the ab plane are similar to those of cellulose I(beta), but the length of the c -axis is nearly twice that of cellulose I. It is proposed that four glucose residues are present in an extended conformation along the c -axis of the unit cell. The data presented show that glycosynthases represent promising enzymic systems for the synthesis of novel polysaccharides with specific and controlled structures, and for the analysis in vitro of the mechanisms of polymerization and crystallization of polysaccharides.
Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer ...the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16–34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites –1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G = Glcβ(1→4) and X = Xylα(1→6)Glcβ(1→4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with kcat/Km values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites –2 and –3 (for backbone Glc residues) and +2′ (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal = Galβ(1→4)) is the best donor substrate with a “blocked” nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.
Plant XETs XG (xyloglucan) endotransglycosylases catalyse the transglycosylation from a XG donor to a XG or low-molecular-mass XG fragment as the acceptor, and are thought to be important enzymes in ...the formation and remodelling of the cellulose-XG three-dimensional network in the primary plant cell wall. Current methods to assay XET activity use the XG polysaccharide as the donor substrate, and present limitations for kinetic and mechanistic studies of XET action due to the polymeric and polydisperse nature of the substrate. A novel activity assay based on HPCE (high performance capillary electrophoresis), in conjunction with a defined low-molecular-mass XGO {XG oligosaccharide; (XXXGXXXG, where G=Glcbeta1,4- and X=Xylalpha1,6Glcbeta1,4-)} as the glycosyl donor and a heptasaccharide derivatized with ANTS 8-aminonaphthalene-1,3,6-trisulphonic acid; (XXXG-ANTS) as the acceptor substrate was developed and validated. The recombinant enzyme PttXET16A from Populus tremula x tremuloides (hybrid aspen) was characterized using the donor/acceptor pair indicated above, for which preparative scale syntheses have been optimized. The low-molecular-mass donor underwent a single transglycosylation reaction to the acceptor substrate under initial-rate conditions, with a pH optimum at 5.0 and maximal activity between 30 and 40 degrees C. Kinetic data are best explained by a ping-pong bi-bi mechanism with substrate inhibition by both donor and acceptor. This is the first assay for XETs using a donor substrate other than polymeric XG, enabling quantitative kinetic analysis of different XGO donors for specificity, and subsite mapping studies of XET enzymes.
Glycosyltransferases (GT) catalyse the biosynthesis of complex carbohydrates which are the most abundant group of molecules in nature. They are involved in several key mechanisms such as cell ...signalling, biofilm formation, host immune system invasion or cell structure and this in both prokaryotic and eukaryotic cells. As a result, research towards complete enzyme mechanisms is valuable to understand and elucidate specific structure-function relationships in this group of molecules. In a next step this knowledge could be used in GT protein engineering, not only for rational drug design but also for multiple biotechnological production processes, such as the biosynthesis of hyaluronan, cellooligosaccharides or chitooligosaccharides.
Generation of these poly- and/or oligosaccharides is possible due to a common feature of several of these GTs: processivity. Enzymatic processivity has the ability to hold on to the growing polymer chain and some of these GTs can even control the number of glycosyl transfers. In a first part, recent advances in understanding the mechanism of various processive enzymes are discussed. To this end, an overview is given of possible engineering strategies for the purpose of new industrial and fundamental applications. In the second part of this review, we focused on specific chain length-controlling mechanisms, i.e., key residues or conserved regions, and this for both eukaryotic and prokaryotic enzymes.
Glycoside hydrolases that release fixed carbon from the plant cell wall are of considerable biological and industrial importance.
These hydrolases contain non-catalytic carbohydrate binding modules ...(CBMs) that, by bringing the appended catalytic domain
into intimate association with its insoluble substrate, greatly potentiate catalysis. Family 6 CBMs (CBM6) are highly unusual
because they contain two distinct clefts (cleft A and cleft B) that potentially can function as binding sites. Henshaw et al. (Henshaw, J., Bolam, D. N., Pires, V. M. R., Czjzek, M., Henrissat, B., Ferreira, L. M. A., Fontes, C. M. G. A., and Gilbert,
H. J. (2003) J. Biol. Chem. 279, 21552-21559) show that Cm CBM6 contains two binding sites that display both similarities and differences in their ligand specificity. Here we report
the crystal structure of Cm CBM6 in complex with a variety of ligands that reveals the structural basis for the ligand specificity displayed by this protein.
In cleft A the two faces of the terminal sugars of β-linked oligosaccharides stack against Trp-92 and Tyr-33, whereas the
rest of the binding cleft is blocked by Glu-20 and Thr-23, residues that are not present in CBM6 proteins that bind to the
internal regions of polysaccharides in cleft A. Cleft B is solvent-exposed and, therefore, able to bind ligands because the
loop, which occludes this region in other CBM6 proteins, is much shorter and flexible (lacks a conserved proline) in Cm CBM6. Subsites 2 and 3 of cleft B accommodate cellobiose (Glc-β-1,4-Glc), subsite 4 will bind only to a β-1,3-linked glucose,
whereas subsite 1 can interact with either a β-1,3- or β-1,4-linked glucose. These different specificities of the subsites
explain how cleft B can accommodate β-1,4-β-1,3- or β-1,3-β-1,4-linked gluco- configured ligands.
Introducción: El objetivo del presente estudio fue analizar la evolución de las necesidades hídricas de un equipo de baloncesto femenino amateur, en situación de partido y durante toda una fase ...eliminatoria, a partir de la pérdida de peso experimentada. Además, se analizó la existencia de posibles diferencias en el nivel de deshidratación en función de la posición de juego.Material y Métodos: Estudio piloto de un solo grupo con mediciones antes de la competición y después de la competición. Jugadoras amateurs de baloncesto (n=10) y sus recipientes de rehidratación fueron pesadas antes y después de los 10 partidos analizados. Las variaciones de peso fueron evaluadas teniendo en cuenta el líquido ingerido de los bidones de reposición y el líquido evacuado a través de la orina.Resultados: Los resultados de este estudio sugieren la existencia de diferencias significativas entre el peso corporal antes y después de cada partido (z=8,551; p<,0005). La magnitud de estas diferencias parece ser muy distinta en función del partido y la jugadora analizada, con valores medios que oscilan entre los 0,63kg (0,9% Peso Corporal) y los 0,95kg (1,37% Peso Corporal) de pérdida de peso corporal. Además, no se observaron diferencias significativas en el nivel de deshidratación en función de la posición de juego (F=1,59; p=0,1929).Conclusiones: Se confirma la existencia de una alta variabilidad intra e interpersonal en cuanto a la pérdida de masa corporal durante los 10 partidos analizados, lo que sugiere la necesidad de una monitorización y rehidratación individualizada.
Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the ...degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture.
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•Mutation of the assisting residue of a GH18 chitinase confers glycosynthase-like activity.•Additional active site mutations significantly enhance glycosynthase efficiency.•Chitin ...oligomers with degree of polymerization ≥10 are produced as insoluble polymers.•The methodology can be extended to partially deacetylated chitosan oligomers/polymers.
Depolymerization of chitin results in chitooligosaccharides (COS) that induce immunostimulatory effects and disease protective responses and have many potential applications in agriculture and medicine. Isolation of bioactive COS with degree of polymerization (DP) larger than six from chitin hydrolyzates is hampered by their water insolubility. Enzymatic synthesis by exploiting the transglycosylation activity of GH18 chitinases offers a potential strategy to access oligomers in the range of bioactive DPs. We engineered SpChiD chitinase as a glycosynthase by mutation of the assisting residue of the catalytic triad in the substrate-assisted mechanism for polymerization of an oxazoline substrate (DP5ox). The insoluble polymer containing DP10 was partially hydrolyzed due to the significant residual hydrolase activity of the mutant enzyme. Combined mutations that strongly reduce the hydrolytic activity, in which the original catalytic triad only retains the essential acid/base residue, together with neighboring mutations in the -1/+1 subsites region, render glycosynthase-like chitinases able to produce chitin oligomers with DP10 as major product in good yields.
Chitin deacetylases (CDAs) emerge as a valuable tool to produce chitosans with a nonrandom distribution of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units. We hypothesized before that CDAs ...tend to bind certain sequences within the substrate matching their subsite preferences for either GlcNAc or GlcN units. Thus, they deacetylate or N-acetylate their substrates at nonrandom positions. To understand the molecular basis of these preferences, we analyzed the binding site of a CDA from Pestalotiopsis sp. (PesCDA) using a detailed activity screening of a site-saturation mutagenesis library. In addition, molecular dynamics simulations were conducted to get an in-depth view of crucial interactions along the binding site. Besides elucidating the function of several amino acids, we were able to show that only 3 residues are responsible for the highly specific binding of PesCDA to oligomeric substrates. The preference to bind a GlcNAc unit at subsite -2 and -1 can mainly be attributed to N75 and H199, respectively. Whereas an exchange of N75 at subsite -2 eliminates enzyme activity, H199 can be substituted with tyrosine to increase the GlcN acceptance at subsite -1. This change in substrate preference not only increases enzyme activity on certain substrates and changes composition of oligomeric products but also significantly changes the pattern of acetylation (PA) when N-acetylating polyglucosamine. Consequently, we could clearly show how subsite preferences influence the PA of chitosans produced with CDAs.
Xyloglucan endo-transglycosylases (XETs) are key enzymes involved in the restructuring of plant cell walls during morphogenesis. As members of glycoside hydrolase family 16 (GH16), XETs are predicted ...to employ the canonical retaining mechanism of glycosyl transfer involving a covalent glycosyl-enzyme intermediate. Here, we report the accumulation and direct observation of such intermediates of PttXET16–34 from hybrid aspen by electrospray mass spectrometry in combination with synthetic “blocked” substrates, which function as glycosyl donors but are incapable of acting as glycosyl acceptors. Thus, GalGXXXGGG and GalGXXXGXXXG react with the wild-type enzyme to yield relatively stable, kinetically competent, covalent GalG-enzyme and GalGXXXG-enzyme complexes, respectively (Gal = Galβ(1→4), G = Glcβ(1→4), and X = Xylα(1→6)Glcβ(1→4)). Quantitation of ratios of protein and saccharide species at pseudo-equilibrium allowed us to estimate the free energy change (ΔG0) for the formation of the covalent GalGXXXG-enzyme as 6.3–8.5 kJ/mol (1.5–2.0 kcal/mol). The data indicate that the free energy of the β(1→4) glucosidic bond in xyloglucans is preserved in the glycosyl-enzyme intermediate and harnessed for religation of the polysaccharide in vivo.
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