β-D-Glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1→3),(1→4)β-D-glucan synthase

Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (14)-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four `U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called `hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species,Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a `class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (14)-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (14)-glycan synthases in plants. For example, the mixed-linkage (13)(14)-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of other -linked cross-linking glycans. In this respect, the enzymatic properties of the mixed-linkage -glucan synthases not only provide special insight into the mechanisms of (14)-glycan synthesis but may also uncover the genes that encode the synthases themselves.     

Curdlan and other bacterial (1→3)-β-d-glucans

Three structural classes of (13)--d-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (13)--glucans and side-chain-branched (13,12)--glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (13,16)--glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (13)--glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (13)--glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.     

Chemical modification of botryosphaeran: structural characterization and anticoagulant activity of a water-soluble sulfonated (1-->3)(1-->6)-β-D-glucan

The exopolysaccharide botryosphaeran (EPS(GLC); a (1--> 3)(1-->6)-β-D-glucan from Botryosphaeria rhodina MAMB- 05) was sulfonated to produce a water-soluble fraction (EPS(GLC)-S) using pyridine and chlorosulfonic acid in formamid. This procedure was then repeated twice to produce another fraction (EPSGLC-RS) with a higher degree of substitution (DS, 1.64). The purity of each botryosphaeran sample (unsulfonated and sulfonated) was assessed by gel filtration chromatography (Sepharose CL-4B), where each polysaccharide was eluted as a single symmetrical peak. The structures of the sulfonated and re-sulfonated botryosphaerans were investigated using ultraviolet-visible (UV-Vis), Fourier-transform infrared (FT-IR), and (13)C nuclear magnetic resonance ((13)C NMR) spectroscopies. EPS(GLC) and EPS(GLC)-RS were also assayed for anticoagulation activity, and EPS(GLC)-RS was identified as an anticoagulant.     

Re-engineering specificity in 1,3-1, 4-β-glucanase to accept branched xyloglucan substrates

Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-β-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-β-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-β-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some β-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-β-glucanase β-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity.     

Relationship between indoor and outdoor airborne fungal spores, pollen, and (1→3)-β-D-glucan in homes without visible mold growth

In this exploratory study, indoor and outdoor airborne fungal spores, pollen, and (1→3)-β-D-glucan levels were determined through long-term sampling (24-h) using a Button Personal Inhalable Aerosol Sampler. The air samples were collected in five Cincinnati area homes that had no visible mold growth. The total count of fungal spores and pollen in the collected samples was conducted under the microscope and Limulus Amebocyte Lysate (LAL) chromogenic assay method was utilized for the determination of the (1→3)-β-D-glucan concentration. For the combined number concentration of fungal spores and pollen, the indoor and outdoor geometric mean values were 573 and 6,435 m⁻³, respectively, with a geometric mean of the Indoor/Outdoor (I/O) ratio of .09. The geometric means of indoor and outdoor (1→3)-β-D-glucan concentrations were .92 and 6.44 ng m⁻³, respectively, with a geometric mean of the I/O ratio equal to .14. The I/O ratio of (1→3)-β-D-glucan concentration was found to be marginally greater than that calculated based on the combined number concentration of fungal spores and pollen. This suggests that (1→3)-β-D-glucan data are affected not only by intact spores and pollen grains but also by the airborne fragments of fungi, pollen, and plant material, which are ignored by traditional enumeration methodologies. Since the (1→3)-β-D-glucan level may elucidate the total exposure to fungal spores, pollen, and fungal fragments, its I/O ratio may be used as a risk marker for mold and pollen exposure in indoor environments.     

Seasonal variation in (1→3)-β-D-glucan levels with health risk assessment and related factors in indoor environments of microbiology laboratories

(1→3)-β-D-glucan causes respiratory airway irritation as a biomarker for biological agents in indoor environments. We evaluated (1→3)-β-D-glucan levels in three microbiology laboratories every month for one year by applying a health risk assessment methodology and determined the associated environmental factors. In total, 192 samples were retrieved using a new, two-stage bioaerosol cyclone (BC 221) sampler. Analysis of (1→3)-β-D-glucan was performed using the kinetic Limulus amebocyte lysate assay. This study revealed that illumination was negatively correlated with (1→3)-β-D-glucan (r = ?0.206, p = 0.04) and that relative humidity and season were positively associated with (1→3)-β-D-glucan (p < 0.05). Among the environmental factors, season (particularly spring) was most significantly associated with (1→3)-β-D-glucan levels. Future studies regarding the relationship between (1→3)-β-D-glucan levels and other biological agents, including fungi, bacteria, endotoxin, and other environmental factors, should be assessed. The hazard quotient in summer is higher than the acceptable level of 1 for children if they stay in laboratories for 12 hours. Inhalation exposure to (1→3)-β-D-glucan by children in microbiology laboratories is thus not safe in summer.     

Combination of induced autolysis and sodium hypochlorite oxidation for the production of Saccharomyces cerevisiae (1 → 3)-β-D-glucan

(1 3)--D-Glucans have received much attention with respect to their biological functions. A novel method to extract (1 3)--D-glucan from Saccharomyces cerevisiae cell wall is proposed in present work, which is based on the combination of induced autolysis and subsequent oxidation of the autolysed cell by sodium hypochlorite to remove undesirable substances. Influences of temperature, pH value and organic solvent on S. cerevisiae FL 1 autolysis were investigated. Results indicated that each factor had its significant effect on induced autolysis and the optimal conditions were 52 °C, pH 5.5 and 1.5% (v/v) ethyl acetate. The kinetic behaviour of the yeast autolytic process under the optimized conditions was further studied. After 36 h of autolysis, 42.0% (w/w) cellular substances were released while the cell wall nearly remained intact. Finally, an ideal glucan yield as high as 22.9% (w/w) was obtained when S. cerevisiae FL 1 was treated by the novel method.     

Synthesis and stability assay of 4-methylumbelliferyl (1→3)-β-D-pentaglucoside

The synthesis and stability of 4-methylumbelliferyl (1 → 3)-β-D-pentaglucoside 3 are described. The (1 → 3)-β-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method. The acid-solubilized (1 → 3)-β-D-oligoglucosides were prepared by partial acid hydrolysis of glucan. The peracetylated (1 → 3)-β-D-pentaglucoside 1 was obtained by isolation of peracetylated (1 → 3)-β-D-oligoglucoside mixture. The peracetylated 4-methylumbelliferyl (1 → 3)-β-D-pentaglucoside 2 was synthesized by treating compound 1 with the 4-methylumbelliferone and a Lewis acid (SnCl₄) catalyst. NaOMe in dry methanol was used for the deacetylation of the blocked derivative, to give the target compound 3 in an overall yield of 35%. Activity assays with β-glucosidase indicated that compound 3 was much more stable than the corresponding pentasaccharide.     

Synthesis and structure–activity relationship of 3β-(4-alkylthio, -methylsulfinyl,and -methylsulfonylphenyl)tropane and 3β-(4-alkylthiophenyl)nortropane derivatives for monoamine transporters

Early studies led to the identification of 3β-(4-methoxyphenyl)tropane-2β-carboxylic acid methyl ester (5) with high affinity at the DAT (IC₅₀ = 6.5 nM) and 5-HTT (K_i = 4.3 nM), while having much less affinity at the NET (K_i = 1110 nM). In the present study, we replaced the 4′-methoxy group of the 3β-phenyl ring with a bioisosteric 4′-methylthio group to give 7a. We also synthesized a number of 3β-(4-alkylthiophenyl)tropanes 7b–e, 3β-(4-methylsulfinylphenyl) and 3β-(4-methylsulfonylphenyl)tropane analogues 7f–h as well as the 3β-(4-alkylthiophenyl)nortropane derivatives 8–11 to further characterize the structure–activity relationship of this type of compound for binding at monoamine transporters. With exception of the 4′-methylsulfonyl analogue 7h, all the tested compounds possessed high binding affinities at the 5-HTT. The K_i values ranged from 0.19 nM to 49 nM. The 3β-(4-methylthiophenyl)tropane 7a and its N-(3-fluoropropyl) analogue 9a and N-allyl analogue 10a are the most selective compounds for the 5-HTT over the NET (NET/5-HTT = 314–364) in the series. However, none of the compounds showed selectivity similar to 5 for both the DAT and 5-HTT relative to the NET. This study provided useful SAR information for rational design of potent and selective monoamine transporter inhibitors.     

Functional and structural characterization of α-(1->2) branching sucrase derived from DSR-E glucansucrase

ΔN₁₂₃-glucan-binding domain-catalytic domain 2 (ΔN₁₂₃-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN₁₂₃-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite −1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN₁₂₃-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN₁₂₃-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN₁₂₃-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.     

Definitive Solution Structures for the 6-Formylated Versions of 1-(βD-Ribofuranosyl)-, 1-(2′-Deoxy-β-D-Ribofuranosyl)-, and 1-β-D-Arabinofuranosyluracil,and of Thymidine

Abstract

ROESY and NOESY NMR spectroscopic analyses of the ribofuranosyl (1a), 2′-deoxyribofuranosyl (1b), and arabinofuranosyl (1c) derivatives of 6-formyluracil in (CD₃)₂SO and D₂O solutions have established that each exclusive 7,05′-cyclic hemiacetal diastereomer of 1a,b and the major 7,O2′-cyclic hemiacetal diastereomer of 1c possess the 7R configuration. In addition, (7R)-1c has been shown to be thermodynamically more stable than (7S)-1c, contrary to our previous indication. A new, higher yielding synthetic route to 1a has been developed, 1b has been obtained for the first time in crystalline form, the route to 1c has been modified to better accommodate large scale preparations, and a new, fourth member of this class, 6-formylthymidine (1d), has been synthesized and its solution structures in (CD₃)₂SO, D₂O, and CD₃OD have been determined. Antitumor and antiviral evaluations of 1a-c have revealed no significant levels of activity.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Cell wall (1 → 3)- and (1 → 3, 1 → 4)-β-glucans during early grain development in rice (Oryza sativa L.)

Immunogold labeling was used to study the distribution of (1 → 3)-β-glucans and (1 → 3, 1 → 4)-β-glucans in the rice grain during cellularization of the endosperm. At approximately 3–5 d after pollination the syncytial endosperm is converted into a cellular tissue by three developmentally distinct types of wall. The initial free-growing anticlinal walls, which compartmentalize the syncytium into open-ended alveoli, are formed in the absence of mitosis and phragmoplasts. This stage is followed by unidirectional (centripetal) growth of the anticlinal walls mediated by adventitious phragmoplasts that form between adjacent interphase nuclei. Finally, the periclinal walls that divide the alveoli are formed in association with centripetally expanding interzonal phragmoplasts following karyokinesis. The second and third types of wall are formed alternately until the endosperm is cellular throughout. All three types of wall that cellularize the endosperm contain (1 → 3)-β-glucans but not (1 → 3, 1 → 4)-β-glucans, whereas cell walls in the surrounding maternal tissues contain considerable amounts of (1 → 3, 1 → 4)-β-glucans with (1 → 3)-β-glucans present only around plasmodesmata. The callosic endosperm walls remain thin and cell plate-like throughout the cellularization process, appearing to exhibit a prolonged juvenile state. Received: 7 January 1997 / Accepted: 11 February 1997     

Synthesis of methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-β-d-galactopyranoside and methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-O-β-d-galactopyranosyl-(1→6)-β-d-galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α-d-galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β-d-galactopyranoside (4) gave a fully acetylated (1→6)-β-d-galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α-d-galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β-d-galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β-d-galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

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.     

Purification and Characterization of endo-(1→4)-β-xylanase fromGeotrichum candidum 3C

A method of purification of endo-( 1 → 4)-β-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid ofGeotrichum candidum 3C, grown for three days, is described. The enzyme, purified 23-fold, had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as the substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30–45°C for 1 h. With xylan from birch wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50°C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10,12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.