Role of (1,3)(1,4) β-glucan in cell walls: Interaction with cellulose.
Kiemle, S. N., Zhang, X., Esker, A. R., Toriz, G., Gatenholm, P. & Cosgrove, D. J. (2014). Biomacromolecules, 15 (5), 1727-1736.
(1,3)(1,4)-β-D-Glucan (mixed-linkage glucan or MLG), a characteristic hemicellulose in primary cell walls of grasses, was investigated to determine both its role in cell walls and its interaction with cellulose and other cell wall polysaccharides in vitro. Binding isotherms showed that MLG adsorption onto microcrystalline cellulose is slow, irreversible, and temperature-dependent. Measurements using quartz crystal microbalance with dissipation monitoring showed that MLG adsorbed irreversibly onto amorphous regenerated cellulose, forming a thick hydrogel. Oligosaccharide profiling using endo-(1,3)(1,4)-β-glucanase indicated that there was no difference in the frequency and distribution of (1,3) and (1,4) links in bound and unbound MLG. The binding of MLG to cellulose was reduced if the cellulose samples were first treated with certain cell wall polysaccharides, such as xyloglucan and glucuronoarabinoxylan. The tethering function of MLG in cell walls was tested by applying endo-(1,3)(1,4)-β-glucanase to wall samples in a constant force extensometer. Cell wall extension was not induced, which indicates that enzyme-accessible MLG does not tether cellulose fibrils into a load-bearing network.
A rhamnogalacturonan lyase in the Clostridium cellulolyticum cellulosome.
Pagès, S., Valette, O., Abdou, L., Bélaïch, A. & Bélaïch, J. P. (2003). Journal of Bacteriology, 185(16), 4727-4733.
Clostridium cellulolyticum secretes large multienzymatic complexes with plant cell wall-degrading activities named cellulosomes. Most of the genes encoding cellulosomal components are located in a large gene cluster: cipC-cel48F-cel8C-cel9G-cel9E-orfX-cel9H-cel9J-man5K-cel9M. Downstream of the cel9M gene, a new open reading frame was discovered and named rgl11Y. Amino acid sequence analysis indicates that this gene encodes a multidomain pectinase, Rgl11Y, containing an N-terminal signal sequence, a catalytic domain belonging to family 11 of the polysaccharide lyases, and a C-terminal dockerin domain. The present report describes the biochemical characterization of a recombinant form of Rgl11Y. Rgl11Y cleaves the α-L-Rha>i>p-(1→4)-α-D-GalpA glycosidic bond in the backbone of rhamnogalacturonan I (RGI) via a β-elimination mechanism. Its specific activity on potato pectic galactan and rhamnogalacturonan was found to be 28 and 3.6 IU/mg, respectively, indicating that Rgl11Y requires galactan decoration of the RGI backbone. The optimal pH of Rgl11Y is 8.5 and calcium is required for its activity. Rgl11Y was shown to be incorporated in the C. cellulolyticum cellulosome through a typical cohesin-dockerin interaction. Rgl11Y from C. cellulolyticum is the first cellulosomal rhamnogalacturonase characterized.
The Inhibitory Effects of a Rhamnogalacturonan Ι (RG-I) Domain from Ginseng Pectin on Galectin-3 and Its Structure-Activity Relationship.
Gao, X., Zhi, Y., Sun, L., Peng, X., Zhang, T., Xue, H., Tai, G. & Zhou, Y. (2013). Journal of Biological Chemistry, 288(47), 33953-33965.
Pectin has been shown to inhibit the actions of galectin-3, a β-galactoside-binding protein associated with cancer progression. The structural features of pectin involved in this activity remain unclear. We investigated the effects of different ginseng pectins on galectin-3 action. The rhamnogalacturonan I-rich pectin fragment, RG-I-4, potently inhibited galectin-3-mediated hemagglutination, cancer cell adhesion and homotypic aggregation, and binding of galectin-3 to T-cells. RG-I-4 specifically bound to the carbohydrate recognition domain of galectin-3 with a dissociation constant of 22.2 nM, which was determined by surface plasmon resonance analysis. The structure-activity relationship of RG-I-4 was investigated by modifying the structure through various enzymatic and chemical methods followed by activity tests. The results showed that (a) galactan side chains were essential to the activity of RG-I-4, whereas arabinan side chains positively or negatively regulated the activity depending on their location within the RG-I-4 molecule. (b) The activity of galactan chain was proportional to its length up to 4 Gal residues and largely unchanged thereafter. (c) The majority of galactan side chains in RG-I-4 were short with low activities. (d) The high activity of RG-I-4 resulted from the cooperative action of these side chains. (e) The backbone of the molecule was very important to RG-I-4 activity, possibly by maintaining a structural conformation of the whole molecule. (f) The isolated backbone could bind galectin-3, which was insensitive to lactose treatment. The novel discovery that the side chains and backbone play distinct roles in regulating RG-I-4 activity is valuable for producing highly active pectin-based galectin-3 inhibitors.
Family 6 carbohydrate‐binding modules display multiple β1,3‐linked glucan‐specific binding interfaces
Correia, M. A. S., Pires, V. M. R., Gilbert, H. J., Bolam, D. N., Fernandes, V. O., Alves, V. D., Prates, J. A. M., Ferreira, L. M. A. & Fontes, C. M. G. (2009). FEMS Microbiology Letters, 300(1), 48-57.
Noncatalytic carbohydrate-binding modules (CBMs), which are found in a variety of carbohydrate-degrading enzymes, have been grouped into sequence-based families. CBMs, by recruiting their appended enzymes onto the surface of the target substrate, potentiate catalysis particularly against insoluble substrates. Family 6 CBMs (CBM6s) display unusual properties in that they present two potential ligand-binding sites termed clefts A and B, respectively. Cleft B is located on the concave surface of the β-sandwich fold while cleft A, the more common binding site, is formed by the loops that connect the inner and the outer β-sheets. Here, we report the biochemical properties of CBM6-1 from Cellvibrio mixtus CmCel5A. The data reveal that CBM6-1 specifically recognizes β1,3-glucans through residues located both in cleft A and in cleft B. In contrast, a previous report showed that a CBM6 derived from a Bacillus halodurans laminarinase binds to β1,3-glucans only in cleft A. These studies reveal a different mechanism by which a highly conserved protein platform can recognize β1,3-glucans.
Biochemical and structural characterization of the intracellular mannanase AaManA of Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase family belonging to clan GH-A.
Zhang, Y., Ju, J., Peng, H., Gao, F., Zhou, C., Zeng, Y., Xue, Y., Li, Y., Henrissat, B., Gao, G. F. & Ma, Y. (2008). Journal of Biological Chemistry, 283(46), 31551-31558.
An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-β-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9Å resolution showed that AaManA adopted a (β/α)8 -barrel fold. Two catalytic residues were identified: Glu151 at the C terminus of β-stand β4 and Glu231 at the C terminus of β7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr95 and Cys150. We propose a model of substrate binding in AaManA in which Glu282 interacts with the axial OH-C(2) in–2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.
Modelling of xyloglucan, pectins and pectic side chains binding onto cellulose microfibrils.
Zykwinska, A., Thibault, J. F. & Ralet, M. C. (2008). Carbohydrate Polymers, 74(1), 23-30.
Binding modelling of tamarind and pea xyloglucans, sugar beet and potato pectins, and pectic side chains (branched arabinan, debranched arabinan, galactan) onto microcrystalline Avicel cellulose and primary cell wall (PCW) cellulose was performed. The most commonly used binding models, namely the Langmuir, the Freundlich and the Scatchard models, were applied to the data. It appeared that the Freundlich model was more appropriate to describe the binding of all the polysaccharides used in this study. The heterogeneity index calculated from the slope of Freundlich isotherms highlights an important heterogeneity of Avicel and PCW cellulose surfaces, in agreement with the Scatchard representation.
Evidence for synergy between family 2b carbohydrate binding modules in Cellulomonas fimi xylanase 11A.
Bolam, D. N., Xie, H., White, P., Simpson, P. J., Hancock, S. M., Williamson, M. P. & Gilbert, H. J. (2001). Biochemistry, 40(8), 2468-2477.
Glycoside hydrolases often contain multiple copies of noncatalytic carbohydrate binding modules (CBMs) from the same or different families. Currently, the functional importance of this complex molecular architecture is unclear. To investigate the role of multiple CBMs in plant cell wall hydrolases, we have determined the polysaccharide binding properties of wild type and various derivatives of Cellulomonas fimi xylanase 11A (Cf Xyn11A). This protein, which binds to both cellulose and xylan, contains two family 2b CBMs that exhibit 70% sequence identity, one internal (CBM2b-1), which has previously been shown to bind specifically to xylan and the other at the C-terminus (CBM2b-2). Biochemical characterization of CBM2b-2 showed that the module bound to insoluble and soluble oat spelt xylan and xylohexaose with Ka values of 5.6 × 104, 1.2 × 104, and 4.8 × 103 M-1, respectively, but exhibited extremely weak affinity for cellohexaose (<102 M-1), and its interaction with insoluble cellulose was too weak to quantify. The CBM did not interact with soluble forms of other plant cell wall polysaccharides. The three-dimensional structure of CBM2b-2 was determined by NMR spectroscopy. The module has a twisted “β-sandwich” architecture, and the two surface exposed tryptophans, Trp 570 and Trp 602, which are in a perpendicular orientation with each other, were shown to be essential for ligand binding. In addition, changing Arg 573 to glycine altered the polysaccharide binding specificity of the module from xylan to cellulose. These data demonstrate that the biochemical properties and tertiary structure of CBM2b-2 and CBM2b-1 are extremely similar. When CBM2b-1 and CBM2b-2 were incorporated into a single polypeptide chain, either in the full-length enzyme or an artificial construct comprising both CBM2bs covalently joined via a flexible linker, there was an approximate 18−20-fold increase in the affinity of the protein for soluble and insoluble xylan, as compared to the individual modules, and a measurable interaction with insoluble acid-swollen cellulose, although the Ka (6.0 × 104 M-1) was still much lower than for insoluble xylan (Ka = 1.0 × 106 M-1). These data demonstrate that the two family 2b CBMs of Cf Xyn11A act in synergy to bind acid swollen cellulose and xylan. We propose that the increased affinity of glycoside hydrolases for polysaccharides, through the synergistic interactions of CBMs, provides an explanation for the duplication of CBMs from the same family in some prokaryotic cellulases and xylanases.
In vitro biosynthesis of 1,4-β-galactan attached to a pectin–xyloglucan complex in pea.
Abdel-Massih, R. M., Baydoun, E. A. H., & Brett, C. T. (2003). Planta, 216(3), 502-511.
Particulate enzyme preparations were prepared from etiolated pea (Pisum sativum L.) epicotyls and used to assay for 1,4-β-galactan synthase using UDP-[U-14C]galactose. Optimum conditions for 1,4-β-galactan synthesis were determined. The enzyme products were characterized by selective enzymic degradation, gel permeation chromatography and anion-exchange chromatography. Evidence was obtained for the formation of 1,4-β-galactan chain attached to a pectic backbone containing both polygalacturonic acid and rhamnogalacturonan I. The results also indicated that part or all of this nascent pectin was present as a complex with xyloglucan.