Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

The Physiology and Molecular Biology of Plant 1,3-β-D-Glucanases and 1,3;1,4-β-D-Glucanases

This review covers the physiology and molecular biology of the plant β-glucanases possessing either endo-1,3-β-D-glucanase (EC 3.2.1.39) or endo-1,3;1,4-β-D-glucanase (EC 3.2.1.73) activity. These β-glucanases are structurally related enzymes that are believed to be involved in many important aspects of plant physiology and development, such as germination, growth, defense against pathogens, flowering, cellular and tissue development and differentiation, and probably other roles. They also are regulated by numerous plant hormones, biotic and abiotic elicitors and stresses, and they exhibit complex tissue- and developmental-specific gene expression.     

（1,3;1,4）-β-D-Glucans in Cell Walls of the Poaceae,Lower Plants,and Fungi： A Tale of Two Linkages

（1,3;1,4）-β-D-Glucans consist of unbranched and unsubstituted chains of （1,3）- and （1,4）-β-glucosyl residues, in which the ratio of （1,4）-β-D-glucosyl residues to （1,3）-β-D-glucosyl residues appears to influence not only the physicochemical properties of the polysaccharide and therefore its functional properties in cell walls, but also its adoption by different plant species during evolution. The （1,3; 1,4）-β-D-glucans are widely distributed as non-cellulosic matrix phase polysaccharides in cell walls of the Poaceae, which evolved relatively recently and consist of the grasses and commercially important cereal species, but they are less commonly found in lower vascular plants, such as the horsetails, in algae and in fungi. The （1,3;1,4）-β-D-glucans have often been considered to be components mainly of primary cell walls, but recent observations indicate that they can also be located in secondary walls of certain tissues. Enzymes involved in the depolymerisation of （1,3;1,4）-β-D-glucans have been well characterized. In contrast, initial difficulties in purifying the enzymes responsible for （1,3;1,4）-β-D-glucan biosynthesis slowed progress in the identification of the genes that encode （1,3;1,4）-β-D-glucan synthases, but emerging comparative genomics and associated techniques have allowed at least some of the genes that contribute to （1,3;1,4）-β-D-glucan synthesis in the Poaceae to be identified. Whether similar genes and enzymes also mediate （1,3;1,4）-β-D-glucan biosynthesis in lower plants and fungi is not yet known. Here, we compare the different fine structures of （1,3;1,4）-β-D-glucans across the plant kingdom, present current information on the genes that have been implicated recently in their biosynthesis, and consider aspects of the cell biology of （1,3;1,4）-β-D-glucan biosynthesis in the Poaceae.     

Cloning and expression of the Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in Escherichia coli

A gene coding for the endo--1,3-1,4-glucanase of B. circulans ATCC21367 was cloned into Escherichia coli. The cloned enzyme hydrolyzed lichenan or barley -glucan to produce 3-O--cellobiosyl-d-glucose as a main product but was inactive with carboxymethyl cellulose, laminarin and xylan. The enzyme, M _r=28 kDa, remained within the cytoplasm of E. coli. A 771 bp open reading frame was in the 2 kb PstI fragment of the recombinant plasmid pLL200K. The deduced protein sequence consists of 257 amino acids and has a putative signal peptide of 26 amino acids. The amino acid sequence of the endo--1,3-1,4-glucanase showed 68 and 51% homology to previously reported endo--1,3-1,4-glucanases from Bacillus strain N-137 and B. brevis, respectively.     

A Mutational Analysis of Killer Toxin Resistance in Saccharomyces Cerevisiae Identifies New Genes Involved in Cell Wall (1 -> 6)-β-Glucan Synthesis

Recessive mutations leading to killer resistance identify the KRE9, KRE10 and KRE11 genes. Mutations in both the KRE9 and KRE11 genes lead to reduced levels of (1 -> 6)-β-glucan in the yeast cell wall. The KRE11 gene encodes a putative 63-kD cytoplasmic protein, and disruption of the KRE11 locus leads to a 50% reduced level of cell wall (1 -> 6)-glucan. Structural analysis of the (1 -> 6)-β-glucan remaining in a kre11 mutant indicates a polymer smaller in size than wild type, but containing a similar proportion of (1 -> 6)- and (1 -> 3)-linkages. Genetic interactions among cells harboring mutations at the KRE11, KRE6 and KRE1 loci indicate lethality of kre11 kre6 double mutants and that kre11 is epistatic to kre1, with both gene products required to produce the mature glucan polymer at wild-type levels. Analysis of these KRE genes should extend knowledge of the β-glucan biosynthetic pathway, and of cell wall synthesis in yeast.     

Induction of the Synthesis of Endo-1,4-β-xylanase and β-Galactosidase in Original and Recombinant Strains of the Fungus Penicillium canescens

The induction of synthesis of the secreted enzymes endo-1,4--xylanase (EC 3.2.1.8) and -galactosidase (EC 3.2.1.23) in original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for induction: the synthesis of -galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4--xylanase was observed at 5–10 mM. An increase in the number of endo-1,4--xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of -galactosidase; the synthesis of endo-1,4--xylanase in the high-copy-number recombinant producing -galactosidase was affected to a lesser extent. The amount of enzymes synthesized did not depend on the saccharide used as the sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.     

Unusual Role of the 3-OH Group of Oligosaccharide Substrates in the Mechanism of Bacillus 1,3-1,4-β-glucanase

Abstract

In the mechanism of retaining β-glycosidases, the 2-hydroxyl group of the substrate in the monosaccharyl unit involved in catalysis (subsite -1) is beleived to play an important role through hydrogen bonding interactions with protein residues that are optimized at the transition state. Commonly, removal of the 2-OH group of the substrate results in a 10–12 kcal·mol^-1 transition state destabilization. However, this effect seems not to be general as reported here for Bacillus 1,3-1,4-β-glucanase, a family 16 retaining endo-glycosidase. A p-nitrophenol 2-deosxy tetrasaccharide substrate was synthesized to probe the involvement of the 2-OH group in catalysis. Comparative kinetics with wild-type and subsite +1 mutants show that the 2-deoxy analog is a better substrate than the corresponding 2-hydroxy substrate. It is tentatively proposed that the 2-deoxy analog adopts a different conformation upon binding that compensates for the lack of the 2-OH substituent.     

A Lichen Lectin Specifically Binds to the α-1,4-Polygalactoside Moiety of Urease Located in the Cell Wall of Homologous Algae

A lectin from the lichen Evernia prunastri developing arginase activity (EC. 3.5.3.1) binds to the homologous algae that contain polygalactosilated urease (EC. 3.5.1.5) in their cell walls acting as a lectin ligand. The enzyme bound to its ligand shows to be inactive to hydrolyze of arginine. Hydrolysis of the galactoside moiety of urease in intact algae with α-1,4-galactosidase (EC. 3.2.1.22) releases high amount of D-galactose and impedes the binding of the lectin to the algal cell wall. However, the use of β-,4-galactosidase (EC.3.2.1.23) releases low amounts of D-galactose from the algal cell wall and does not change the pattern of binding of the lectin to its ligand. The production of glycosilated urease is restricted to the season in which algal cells divide and this assures the recognition of new phycobiont produced after cell division by its fungal partner.Key Words: arginase, cell wall, evernia prunastri, lectin ligand, phycobiont, urease     

Prognostic Potential of the Panfungal Marker (1 → 3)-β-d-Glucan in Invasive Mycoses Patients

Mycopathologia - We analyze the prognostic potential of (1?→?3)-β-d-glucan (BG) levels in predicting clinical outcomes in patients with invasive fungal infections, on a...     

1,3-β-d-Glucan synthase ofNeurospora crassa: Partial purification and characterization of solubilized enzyme activity

1,3-β-d-Glucan synthase activity ofNeurospora crassa was rendered soluble by treatment of crude protoplast lysates with 0.1% 3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 1 mM dithiothreitol, 200 mM inorganic phosphate, 10 μM GTP, and 600 mM glycerol. Approximately 50% of enzyme activity was solubilized; soluble enzyme activity was purified 5.6-fold with a net 38% recovery by sucrose gradient density sedimentation. Partially purified enzyme activity had a half-life of 60 ± 10 h at 4°C, aK_m,app of 0.75 ± 0.05 mM, and a V_max,app of 35 ± 1 enzyme units/mg protein.     

Study of the influence of temperature on the dynamics of the catalytic cleft in 1,3-1,4-β-glucanase by molecular dynamics simulations

The dependence of some molecular motions in the enzyme 1,3-1,4-β-glucanase from Bacillus licheniformis on temperature changes and the role of the calcium ion in them were explored. For this purpose, two molecular dynamics simulated trajectories along 4 ns at low (300 K) and high (325 K) temperatures were generated by the GROMOS96 package. Several structural and thermodynamic parameters were calculated, including entropy values, solvation energies, and essential dynamics (ED). In addition, thermoinactivation experiments to study the influence of the calcium ion and some residues on the activity were conducted. The results showed the release of the calcium ion, which, in turn, significantly affected the movements of loops 1, 2, and 3, as shown by essential dynamics. These movements differ at low and high temperatures and affect dramatically the activity of the enzyme, as observed by thermoinactivation studies. The first two authors contributed equally to this work     

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)₄ complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)₂ lobes joined with D₂ symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)₄ complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)₄ kinase core, because a β₄ subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.     

Construction of a highly thermostable 1,3-1,4-β-glucanase by combinational mutagenesis and its potential application in the brewing industry

Objectives

To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-β-glucanase by combinational mutagenesis and to test its effect in congress mashing.

Results

A mutant β-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T ₅₀ value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k _cat value were 42,734 U mg^?1 and 189 s^?1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml^?1, which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively.

Conclusions

The mutant β-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.