Positional isomers of thioxylobiose, their synthesis and inducing ability for D-xylan-degrading enzymes in the yeast Cryptococcus albidus.

Isomeric S-linked 2-thioxylobiose 10, 3-thioxylobiose 17, and 4-thioxylobiose 19 were conveniently prepared by SN2 displacement of suitable triflylglycoses with the sodium salt of 2,3,4-tri-O-acetyl-1-thio-beta-D-glucopyranose, either in N,N-dimethylformamide, or in oxolan in the presence of a sodium complexing agent. Allyl 3,5-O-isopropylidene-2-O-trifluoromethanesulfonyl-beta-D-lyxofu ranoside was a convenient electrophilic precursor for 10, which was smoothly obtained after a short sequence of deprotection involving conversion to the 1-propenyl glycoside. 1,2:5,6-Di-O-isopropylidene-3-O-trifluoromethylsulfonyl-alpha-D-++ +allofuranose and 1,2,3-tri-O-benzoyl-4-O-trifluoromethylsulfonyl-beta-L-arabinop yranose were the respective precursors for 17 and 19. 4-Thioxylobiose has a highly stimulatory effect on the synthesis of enzymes of the xylanolytic system in the yeast Cryptococcus albidus when applied to the cells in the presence of the natural disaccharide inducer (1----4)-beta-D-xylobiose.     

The alpha-glucuronidase,GlcA67A,of Cellvibrio japonicus utilizes the carboxylate and methyl groups of aldobiouronic acid as important substrate recognition determinants

alpha-Glucuronidases are key components of the ensemble of enzymes that degrade the plant cell wall. They hydrolyze the alpha1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid (4-O-MeGlcA) and the xylan or xylooligosaccharide backbone. Here we report the crystal structure of an inactive mutant (E292A) of the alpha-glucuronidase, GlcA67A, from Cellvibrio japonicus in complex with its substrate. The data show that the 4-O-methyl group of the substrate is accommodated within a hydrophobic sheath flanked by Val-210 and Trp-160, whereas the carboxylate moiety is located within a positively charged region of the substrate-binding pocket. The carboxylate side chains of Glu-393 and Asp-365, on the "beta-face" of 4-O-MeGlcA, form hydrogen bonds with a water molecule that is perfectly positioned to mount a nucleophilic attack at the anomeric carbon of the target glycosidic bond, providing further support for the view that, singly or together, these amino acids function as the catalytic base. The capacity of reaction products and product analogues to inhibit GlcA67A shows that the 4-O-methyl group, the carboxylate, and the xylose sugar of aldobiouronic acid all play an important role in substrate binding. Site-directed mutagenesis informed by the crystal structure of enzyme-ligand complexes was used to probe the importance of highly conserved residues at the active site of GlcA67A. The biochemical properties of K288A, R325A, and K360A show that a constellation of three basic amino acids (Lys-288, Arg-325, and Lys-360) plays a critical role in binding the carboxylate moiety of 4-O-MeGlcA. Disruption of the apolar nature of the pocket created by Val-210 (V210N and V210S) has a detrimental effect on substrate binding, although the reduction in affinity is not reflected by an inability to accommodate the 4-O-methyl group. Replacing the two tryptophan residues that stack against the sugar rings of the substrate with alanine (W160A and W543A) greatly reduced activity.     

A novel family of hemicellulolytic α-glucuronidase

Investigation of the xylanolytic enzyme system of the xylose-fermenting yeast Pichia stipitis resulted in the discovery of an extracellular α-glucuronidase efficiently debranching hardwood glucuronoxylan. This activity is not exhibited by more extensively investigated α-glucuronidases of glycoside hydrolase (GH) family 67, operating on substrates in which the uronic acid is linked to the non-reducing xylopyranosyl residues of main chain fragments. The N-terminus of the purified enzyme corresponded exactly to the P. stipitis gene ABN67901 coding for a protein of unknown function. BLAST search revealed the presence of similar genes in genomes of other microorganisms. These results lead to the emergence of a new family of α-glucuronidases.     

Preparation of regioselectively feruloylated p-nitrophenyl α-l-arabinofuranosides and β-d-xylopyranosides—convenient substrates for study of feruloyl esterase specificity

p-Nitrophenyl α-l-arabinofuranoside and β-d-xylopyranoside mono-O-ferulates were prepared by 4-O-acetylferuloylation of corresponding enzymatically prepared di-O-acetates followed by deacetylation. An alternative mild acylation catalysed by zinc oxide was tested on xylopyranoside derivatives. The chemoselective methanolysis of the acetyl groups using neutral catalyst dibutyltin oxide at reflux was used as deacetylation method. Under these conditions a significant feruloyl migration was observed mainly on p-nitrophenyl 3-O-feruloyl-β-d-xylopyranoside resulting in low yields of the positional isomers. Investigation of substrate and positional specificity of different types of feruloyl esterases on the presented compounds in enzyme-coupled assays was reported previously.     

Transacetylations to carbohydrates catalyzed by acetylxylan esterase in the presence of organic solvent

Various conditions were applied to test the ability of acetylxylan esterase (AcXE) from Schizophyllum commune to catalyze acetyl group transfer to methyl beta-D-xylopyranoside (Me-beta-Xylp) and other carbohydrates. The best performance of the enzyme was observed in an n-hexane-vinyl acetate-sodium dioctylsulfosuccinate (DOSS)-water microemulsion at a molar water-detergent ratio (w(0)) of about 4-5. Although the enzyme was found to have a half-life of about 1 h in the system, more than 60% conversion of Me-beta-Xylp to acetylated derivatives was achieved. Under identical reaction conditions, the enzyme acetylated other carbohydrates such as methyl beta-D-cellobioside (Me-beta-Cel), cellotetraose, methyl beta-D-glucopyranoside (Me-beta-Glcp), 2-deoxy-D-glucose, D-mannose, beta-1,4-mannobiose, -mannopentaose, -mannohexaose, beta-1,4-xylobiose and -xylopentaose. This work is the first example of reverse reactions by an acetylxylan esterase and a carbohydrate esterase belonging to family 1.     

Purification and characterization of two forms of endo-beta-1,4-mannanase from a thermotolerant fungus, Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749)

Two extracellular endo-beta-1,4-mannanases, MAN I (major form) and MAN II (minor form), were purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Molecular weights of MAN I and MAN II estimated by SDS-PAGE were 60 and 63 kDa, respectively. IEF afforded several glycoprotein bands with pI values in the range of 4.9-5.2 for MAN I and 4.75-4.9 for MAN II, each exhibiting enzyme activity. MAN I as well as MAN II showed highest activity at pH 4.5 and 60 degrees C and were stable in the pH range 4.5-8.5 and up to 55 degrees C. In accordance with the ability of the enzymes to catalyze transglycosylation reactions, 1H NMR spectroscopy of reaction products generated from mannopentaitol confirmed the retaining character of both enzymes. Both MAN I and MAN II exhibited essentially identical kinetic parameters for polysaccharides and a similar hydrolysis pattern of various oligomeric and polymeric substrates. Both beta-mannanases contained identical internal amino acid sequence corresponding to glycoside hydrolase family 5 and also a cellulose-binding module. These data suggested that both MAN I and MAN II are products of the same gene differing in posttranslational modification. Indeed, the corresponding gene was identified within the recently sequenced Aspergillus fumigatus genome (http://sanger.ac.uk/Projects/A_fumigatus/).     

Specificity of cellulase and β-xylanase induction in Trichoderma reesei QM 9414

Cellulose- and xylan-degrading enzymes of Trichoderma reesei QM 9414 induced by, sophorose, xylobiose, cellulose and xylan were analyzed by isoelectric focusing. The sophorose-induced enzyme system contained two types of endo-1,4--glucanases (EC 3.2.1.4), one specific for cellulose and the other non-specific, hydrolyzing both cellulose and xylan, and exo-1,4--glucanases (cellobiohydrolases I, EC 3.2.1.91), i.e. all types of glucanases that are produced during growth on cellulose. Specific endo-1,4--xylanases (EC 3.2.1.8) present in the cellulose-containing medium were less abundant in the sophorose-induced enzyme system. Xylobiose and xylan induced only specific endo-1,4--xylanases. It is concluded that syntheses of cellulases and -xylanases in T. reesei QM 9414 are under separate control and that the non-specific endo-1,4--glucanases are constituents of the cellulose-degrading enzyme system.     

Lysis of Saccharomyces cerevisiae with 2-deoxy-2-fluoro-D-glucose, an inhibitor of the cell wall glucan synthesis

The effect of a synthetic glucose analogue, 2-deoxy-2-fluoro-d-glucose (FG) on growth and glucose metabolism of Saccharomyces cerevisiae was studied. The addition of FG (0.005-0.05%) to a 2% glucose medium resulted in reduction of the initial growth rate and, after several hours, in a complete cessation of the culture growth. These two events were due to extensive lysis of the population which continued long after the period when no more growth was recorded. Electron microscope examination of lysed cells showed that the lysis was a consequence of a dissolution of the cell walls. FG inhibited to a similar extent the initial growth rate and the incorporation of radioactivity from labeled glucose into growing population. The inhibition of radioactivity incorporation from glucose by growing protoplasts was much less. The yeast was found to be extremely FG sensitive whenever the synthesis of new cell wall material was involved. All observations imply that FG interferes mainly with the cell wall formation of S. cerevisiae. A comparison of the FG effects on metabolic activity of protoplasts, simultaneous secretion of mannan-proteins into the growth medium, and the formation of glucan fibrils on the surface of protoplasts demonstrated that the cell wall glucan synthesis is the most FG-sensitive process and evidently the growth-limiting factor in intact cells. FG-resistant cells were selected during growth experiments. They exhibited an altered mode of cell division when grown in the presence of FG.     

Substrate and positional specificity of feruloyl esterases for monoferuloylated and monoacetylated 4-nitrophenyl glycosides

4-Nitrophenyl glycosides of 2-, 3-, and 5-O-(E)-feruloyl- and 2- and 5-O-acetyl-alpha-L-arabinofuranosides and of 2-, 3-, and 4-O-(E)-feruloyl- and 2-, 3- and 4-O-acetyl-beta-D-xylopyranosides, compounds mimicking natural substrates, were used to investigate substrate and positional specificity of type-A, -B, and -C feruloyl esterases. All the feruloyl esterases behave as true feruloyl esterases showing negligible activity on sugar acetates. Type-A enzymes, represented by AnFaeA from Aspergillus niger and FoFaeII from Fusarium oxysporum, are specialized for deferuloylation of primary hydroxyl groups, with a very strong preference for hydrolyzing 5-O-feruloyl-alpha-L-arabinofuranoside. On the contrary, type-B and -C feruloyl esterases, represented by FoFaeI from F. oxysporum and TsFaeC from Talaromyces stipitatus, acted on almost all ferulates with exception of 4- and 3-O-feruloyl-beta-D-xylopyranoside. 5-O-Feruloyl-alpha-L-arabinofuranoside was the best substrate for both TsFaeC and FoFaeI, although catalytic efficiency of the latter enzyme toward 2-O-feruloyl-alpha-L-arabinofuranoside was comparable. In comparison with acetates, the corresponding ferulates served as poor substrates for the carbohydrate esterase family 1 feruloyl esterase from Aspergillus oryzae. The enzyme hydrolyzed all alpha-L-arabinofuranoside and beta-D-xylopyranoside acetates. It behaved as a non-specific acetyl esterase rather than a feruloyl esterase, with a preference for 2-O-acetyl-beta-D-xylopyranoside.