Crystallization of alpha-galactosidase from Trichoderma reesei.

An extracellular alpha-galactosidase was isolated from fungus Trichoderma reesei. The purified enzyme had a molecular weight of 54,000 Da and an isoelectric point of 5.25. Crystals of alpha-galactosidase were obtained from a polyethylene glycol 4000 solution by the hanging-drop method. Seeding was used for enlargement of the crystal size. The crystals belong to the orthorhombic space group P2(1)2(1)2 with cell dimensions a = 122.2 A, b = 81.0 A c = 49.7 A and diffract beyond 3.0 A resolution.

Crystals of beta-xylanase from Aspergillus oryzae.

An endo-xylanase was isolated from the culture of fungus Aspergillus oryzae variant D5. The purified enzyme had a molecular weight of 24,000 and the isoelectric point of 3.6. Xylanase crystals were obtained from a polyethylene glycol 6000 solution by the hanging-drop method. Seeding was used for the enlargement of the crystal size. Crystals belong to the monoclinic space group P2(1) with cell dimensions a = 54.9 A, b = 74.5 A, c = 50.8 A, and beta = 108.7 degrees. Crystals diffract beyond 2.5 A resolution.

Crystallization and preliminary X-ray study of minor glucoamylase from Aspergillus awamori variant X-100/D27.

Crystals of the reduced form of glucoamylase were obtained from polyethylene glycol 6000 solution by the hanging-drop method. The protein was treated with alpha-mannosidase to partly remove the sugar component. The crystals belong to the space group P2(1)2(1)2(1) with cell dimensions a = 116.7 A, b = 104.3 A, c = 48.5 A and diffract beyond 2.5 A resolution.

Crystal structure of exo-inulinase from Aspergillus awamori: the enzyme fold and structural determinants of substrate recognition.

Exo-inulinases hydrolyze terminal, non-reducing 2,1-linked and 2,6-linked beta-d-fructofuranose residues in inulin, levan and sucrose releasing beta-d-fructose. We present the X-ray structure at 1.55A resolution of exo-inulinase from Aspergillus awamori, a member of glycoside hydrolase family 32, solved by single isomorphous replacement with the anomalous scattering method using the heavy-atom sites derived from a quick cryo-soaking technique. The tertiary structure of this enzyme folds into two domains: the N-terminal catalytic domain of an unusual five-bladed beta-propeller fold and the C-terminal domain folded into a beta-sandwich-like structure. Its structural architecture is very similar to that of another member of glycoside hydrolase family 32, invertase (beta-fructosidase) from Thermotoga maritima, determined recently by X-ray crystallography The exo-inulinase is a glycoprotein containing five N-linked oligosaccharides. Two crystal forms obtained under similar crystallization conditions differ by the degree of protein glycosylation. The X-ray structure of the enzyme:fructose complex, at a resolution of 1.87A, reveals two catalytically important residues: Asp41 and Glu241, a nucleophile and a catalytic acid/base, respectively. The distance between the side-chains of these residues is consistent with a double displacement mechanism of reaction. Asp189, which is part of the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition.

Crystal structures of beta-galactosidase from Penicillium sp. and its complex with galactose.

Beta-galactosidases catalyze the hydrolysis of beta(1-3) and beta(1-4) galactosyl bonds in oligosaccharides as well as the inverse reaction of enzymatic condensation and transglycosylation. Here we report the crystallographic structures of Penicillium sp. beta-galactosidase and its complex with galactose solved by the SIRAS quick cryo-soaking technique at 1.90 A and 2.10 A resolution, respectively. The amino acid sequence of this 120 kDa protein was first assigned putatively on the basis of inspection of the experimental electron density maps and then determined by nucleotide sequence analysis. Primary structure alignments reveal that Penicillium sp. beta-galactosidase belongs to family 35 of glycosyl hydrolases (GHF-35). This model is the first 3D structure for a member of GHF-35. Five distinct domains which comprise the structure are assembled in a way previously unobserved for beta-galactosidases. Superposition of this complex with other beta-galactosidase complexes from several hydrolase families allowed the identification of residue Glu200 as the proton donor and residue Glu299 as the nucleophile involved in catalysis. Penicillium sp. beta-galactosidase is a glycoprotein containing seven N-linked oligosaccharide chains and is the only structure of a glycosylated beta-galactosidase described to date.

Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism.

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.

Effect of modification of carbohydrate component on properties of glucoamylase.

In this study, we investigated enzymatic deglycosylation of glucoamylase from Aspergillus awamori X 100/D27, a glycoprotein which has two N-linked and about forty short mannose-bearing O-linked sugars per molecule. O-Linked sugars were modified by treatment with alpha-mannosidase and N-linked sugars were removed using endo-beta-N-acetylglucosaminidase F. Analysis of conformational changes following deglycosylation suggests that O-linked sugars essentially contribute to the stabilization of glucoamylase domains. Modification of the carbohydrate component by adding 1-deoxymannojirimycin to the culture medium induced inhibition of alpha-mannosidases involved in the processing, leading to a more complete glycosylation and, consequently, to a higher stability of the enzyme.

Amylolytic activity of IgG and sIgA immunoglobulins from human milk.

BACKGROUND: New natural amylolytic abzymes (Abs) for catalytically active antibodies from human milk have been identified and investigated. METHODS: The amylolytic activity of human milk autoantibodies was studied by TLC and HPLC techniques analyzing the hydrolysis of maltooligosaccharides with different degrees of polymerization and of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside (EPS). IgG and sIgA fractions were isolated from human milk by affinity chromatography. After SDS-PAGE preparation of native IgG and sIgA and their renaturation, the amylolytic activity was in-gel assayed. RESULTS: All electrophoretically homogeneous preparations of IgG and its Fab fragments as well as sIgA antibodies possessed alpha-amylolytic activity. The specific activities of these catalytic antibodies varied in the range from 1.83 up to 3.33 kat/kg, which is about one order of magnitude higher than that for IgGs from the sera of cancer patients. IgG and sIgA fractions showed Michaelis constants for hydrolysis of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside in the range of 10(-4) M/l. Fractions of autoantibodies from different donors exhibited different modes of action in hydrolysis of maltooligosaccharides, maltose and p-nitrophenyl-alpha-D-glucopyranose. CONCLUSIONS: IgG antibodies, their Fab fragments, and sIgA fractions isolated from human milk of healthy women possessed amylolytic activity in the hydrolysis of maltooligosaccharides and several artificial substrates.

Transglycosylation activity of alpha-D-galactosidase from Trichoderma reesei. An investigation of the active site.

The transglycosylation reaction catalyzed by alpha-D-galactosidase from the mycelial fungus Trichoderma reesei was studied using p-nitrophenyl alpha-D-galactopyranoside (PNPG). An aliphatic alcohol or the substrate itself can be an acceptor of the galactose residue in this reaction. The transglycosylation products were identified as alkyl galactosides in the case of alcohols or as galactobioside and galactotrioside in the case of PNPG. The transglycosylation rates follow a first-order equation with respect to the alcohol concentrations except for methanol. Affinities of some substrates were estimated from their Ki values in the reaction of the enzyme with PNPG. Transglycosylation of the substrate suggests a model for the enzyme active center. It is proposed that the active center includes two galactose-binding sites and a hydrophobic site.

Microheterogeneity in O-type sugar chains of carbohydrases secreted by Asp. awamori.

This paper deals with microheterogeneity in the structure of O-linked sugars of carbohydrases secreted by Asp. awamori, namely glucoamylase, alpha-galactosidase and alpha-glucosidase. Microheterogeneity was found to be related both to post-secretion deglycosylation and to changes in transferase activity induced by the differences in culturing conditions.

[Cleavage of O-glycosyl bonds in glycopeptides]. / Rasshchepleniia O-glikozil'nykh sviazei v glikopeptidakh.

The possibility of cleavage of the alpha-bond between mannose (or galactose) and serine (or threonine) in the presence of alpha-mannosidase and alpha-galactosidase has been studied. Using model compounds simulating the O-glycosyl bond in glycoproteins, several glycopeptides have been synthesized: N-tertbutyloxycarbonyl-O-alpha-mannopyranosyl-seryl-glycine methylamide (alpha-Man-Ser-Gly), tertbutyl-oxycarbonyl-O-alpha-mannopyranosyl-threonyl- glycine methylamide (alpha-Man-Thr-Gly), N-tertbutyloxy-carbonyl-O-alpha-galactopyranosyl-seryl-glycine methylamide (alpha-Gal-Ser-Gly) as well as N-tertbutyloxy-carbonyl-O-beta-mannopyranosyl-seryl-glycine methylamide (beta-Man-Ser-Gly). The cleavage has been shown to occur in glucoamylase after proteolytic degradation.

A model for cleavage of O-glycosidic bonds in glycoproteins.

The present work investigated the possibility of cleavage of alpha-linkages between mannose or galactose and serine/threonine residues by alpha-mannosidase and alpha-galactosidase. The study was carried out initially with model synthetic compounds imitating the O-glycosidic bond in glycoproteins, and further with glucoamylase. It was shown that alpha-mannosidase and alpha-galactosidase can hydrolyse these linkages after proteolytic digestion of glucoamylase.

Adsorption of glucoamylase from Asp. awamori X-100/D27 on cell walls.

Upon secretion, the major form of glucoamylase from Asp. awamori adsorbs on fungal cell walls. Its substrate binding site showing affinity for alpha-1-4 and alpha-1-6 wall glucans is responsible for adsorption. The minor form in which this site is absent has no sorption ability for cell walls.

Purification, crystallization and preliminary diffraction study of beta-galactosidase from Penicillium sp.

Crystals of an extracellular beta-galactosidase from Penicillium sp. (MW = 120 +/- 5 kDa) have been obtained from a sodium phosphate buffer using PEG as precipitant. The crystals belong to the tetragonal space group P4(1)or P4(3), with unit-cell parameters a = b = 110.82, c = 161.28 A, and diffract to 1.85 A resolution at a synchrotron source.

Purification, crystallization and preliminary X-ray study of beta-xylosidase from Trichoderma reesei.

An extracellular multifunctional beta-xylosidase was purified from a culture of the fungus Trichoderma reesei. The active 95 +/- 5 kDa enzyme has been crystallized from sodium acetate buffer using PEG as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.75, b = 98.54, c = 227.25 A, and diffract beyond 2.7 A resolution. X-ray data were collected from frozen crystals on a synchrotron source.

NMR spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9-15-ppm range.

1H-NMR spectra have been recorded for glucoamylases I and II from Aspergillus awamori var. X100 and from A. niger in the 9-15-ppm region. At least 17 distinct peaks, many of them arising from single protons, are observed. These are designated A-Q, A being the furthest downfield. At least 9 of these are lost rapidly by exchange when the enzyme is placed in D2O. Peaks A, B, E and H undergo distinct shifts with pH change in the pH region 3-7. Several others undergo smaller shifts. Small differences are also seen between the enzymes from the two different sources. Binding of the pseudotetrasaccharide inhibitor acarbose leads to a 0.50-ppm downfield shift of peak B, other smaller changes, and retention of two additional protons in D2O. delta-D-gluconolactone induces shifts in peaks E, H, and L. The slow substrate maltitol causes peak A to broaden and shift, peaks J and K to shift and a new or greatly shifted resonance to appear at 15.4 ppm. It disappears as the maltitol is hydrolyzed. Treatment with iodoacetamide or diethyl pyrocarbonate leads to disappearance of peak D at 12.3 ppm. When this peak was irradiated strong nuclear Overhauser effects (NOE) were observed at 8.01 ppm and 7.22 ppm, positions expected for the C epsilon 1 and C delta 2 protons of an uncharged imidazole ring. We identify D as arising from the N epsilon 2 proton of His254 which is uncharged except at the lowest pH values. Other NOE and two-dimensional NOE spectra have provided additional information. Three mutant forms of the A. niger enzyme, in which tryptophan residues have been replaced by phenylalanine, have been examined. Because of shifts induced by changes in ring current and other environmental effects it is hard to make a direct identification of the resonances from the replaced indole NH protons. However, on the basis of a distinct NOE between peaks E and H we have identified these resonances as arising from the indole NH protons of Trp52 and Trp120. Other possible assignments are considered. The NMR spectra of the glucoamylases I, which have a starch binding domain of about 104 residues at the carboxyl terminus, show four sharp resonances in the 9.7-10.6-ppm range that are not present in the glucoamylases II, which lack this domain. These resonances no doubt represent the four indole NH ring protons from Trp543, Trp562, Trp590 and Trp615. Three of these are very sharp suggesting a high mobility of this domain.

Cloning of a gluconate/polyol dehydrogenase gene from Gluconobacter suboxydans IFO 12528, characterisation of the enzyme and its use for the production of 5-ketogluconate in a recombinant Escherichia coli strain.

A 5-ketogluconate (5-KGA)-forming membrane quinoprotein, gluconate dehydrogenase, was isolated from Gluconobacter suboxydans strain IFO 12528 and partially sequenced. Partial sequences of five internal tryptic peptides were elucidated by mass spectrometry and used to isolate the two adjacent genes encoding the enzyme (EBI accession no. AJ577472). These genes share close homology with sorbitol dehydrogenase from another strain of G. suboxydans (IFO 3255). Substrate specificity of gluconate 5-dehydrogenase (GA 5-DH) turned out to be quite broad, covering many polyols, amino derivatives of carbohydrates, and simple secondary alcohols. There is a broad correlation between the substrate specificity of GA 5-DH and the empirical Bertrand-Hudson rule that predicts the specificity of oxidation of polyols by acetic acid bacteria. Escherichia coli transformed with the genes encoding gluconate dehydrogenase were able to convert gluconic acid into 5-KGA at 75% yield. Furthermore, it was found that 5-KGA can be converted into tartaric acid semialdehyde by a transketolase. These results provide a basis for designing a direct fermentation-based process for conversion of glucose into tartaric acid.

Role of methionine in the active site of alpha-galactosidase from Trichoderma reesei.

alpha-Galactosidase from Trichoderma reesei when treated with H2O2 shows a 12-fold increase in activity towards p-nitrophenyl alpha-D-galactopyranoside. A similar effect is produced by the treatment of alpha-galactosidase with other non-specific oxidants: NaIO4, KMnO4 and K4S4O8. In addition to the increase in activity, the Michaelis constant rises from 0.2 to 1.4 mM, the temperature coefficient decreases by a factor of 1.5 and the pH-activity curve falls off sharply with increasing pH. Galactose (a competitive inhibitor of alpha-galactosidase; Ki 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative activation of the enzyme, because the observed activity changes are related to oxidation of the catalytically important methionine in the active site. NMR measurements and amino acid analysis show that oxidation to methionine sulphoxide of one of five methionines is sufficient to activate alpha-galactosidase. Binding of galactose prevents this. Oxidative activation does not lead to conversion of other H2O2-sensitive amino acid residues, such as histidine, tyrosine, tryptophan and cysteine. The catalytically important cysteine thiol group is quantitatively titrated after protein oxidative activation. Further oxidation of methionines (up to four of five residues) can be achieved by increasing the oxidation time and/or by prior denaturation of the protein. Obviously, a methionine located in the active site of alpha-galactosidase is more accessible. The oxidative-activation phenomenon can be explained by a conformational change in the active site as a result of conversion of non-polar methionine into polar methionine sulphoxide.

Crystallization and preliminary X-ray study of beta--mannosidase from Trichoderma reesei.

beta-Mannosidase from Trichoderma reesei, a 105 kDa glycoprotein, has been crystallized. The crystals belong to the space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell dimensions a = b = 165.86, c = 122.46 A, and diffract beyond 2.75 A resolution. X-ray diffraction data were collected from a frozen crystal on a synchrotron X-ray source.

The action of alpha-mannosidase from Oerskovia sp. on the mannose-rich O-linked sugar chains of glycoproteins.

Alpha-mannosidase was isolated from the culture liquid of Oerskovia sp. The purified enzyme had a molecular mass of 480 kDa and comprises four identical subunits. The enzyme cleaves bonds in side chains of yeast mannan (Km = 0.08 mM, k(cat) = 1.02 micromol x min(-1) x mg(-1)) and reveals a low activity towards p-nitrophenyl alpha-D-mannopyranoside. The alpha-mannosidase is a Ca2+-dependent enzyme and is inhibited by EDTA. The enzyme possess no endo-mannosidase activity releasing only mannose in the reaction with the inversion of anomeric configuration and could be classified as exo-alpha-mannanase. The enzyme revealed a high deglycosylating activity towards the short mannose-rich O-linked carbohydrate chains of glycoproteins.