A sialidase complex from chicken liver: Characterization of a multienzyme complex with β-galactosidase and carboxypeptidase

Mammalian lysosomal sialidase exists as an enzyme complex with β-galactosidase and carboxypeptidase, so-called “protective protein.” In this article, we report that chicken sialidase also occurs as a complex with β-galactosidase and protective protein. The purified sialidase complex had a molecular weight > 700 kDa on gel filtration and showed four protein components of 76, 65, 54 and 48 kDa on SDS-PAGE under nonreducing conditions. N-Terminal sequences of the 65- and 48-kDa proteins were homologous to human lysosomal β-galactosidase and protective protein precursor, respectively. The purified sialidase complex also had carboxypeptidase activity. Both sialidase and carboxypeptidase activities were precipitated together by an antibody against chicken β-galactosidase. The complex reversibly dissociated into 120-kDa β-galactosidase dimer and 100-kDa carboxypeptidase dimer at pH 7.5, but the sialidase irreversibly inactivated during the depolymerization. These findings indicate that chicken sialidase exists as a multienzyme complex, by which the sialidase activity appears to be stabilized.     

Crystal structures of the apo form of β-fructofuranosidase from Bifidobacterium longum and its complex with fructose

We solved the 1.8 ? crystal structure of β-fructofuranosidase from Bifidobacterium longum KN29.1 - a unique enzyme that allows these probiotic bacteria to function in the human digestive system. The sequence of β-fructofuranosidase classifies it as belonging to the glycoside hydrolase family 32 (GH32). GH32 enzymes show a wide range of substrate specificity and different functions in various organisms. All enzymes from this family share a similar fold, containing two domains: an N-terminal five-bladed β-propeller and a C-terminal β-sandwich module. The active site is located in the centre of the β-propeller domain, in the bottom of a 'funnel'. The binding site, -1, responsible for tight fructose binding, is highly conserved among the GH32 enzymes. Bifidobacterium longum KN29.1 β-fructofuranosidase has a 35-residue elongation of the N-terminus containing a five-turn α-helix, which distinguishes it from the other known members of the GH32 family. This new structural element could be one of the functional modifications of the enzyme that allows the bacteria to act in a human digestive system. We also solved the 1.8 ? crystal structure of the β-fructofuranosidase complex with β-D-fructose, a hydrolysis product obtained by soaking apo crystal in raffinose.     

Characterisation and properties of the inclusion complex of 24-epibrassinolide with β-cyclodextrin

This paper reports the first study of an inclusion complex of abrassinosteroid with -cyclodextrin. The formation of inclusion complexesbetween 24-epibrassinolide and -cyclodextrin was confirmed by theirphysicochemical properties and the compounds were analysed by differentialscanning calorimetry, powder X-ray diffraction, nuclear magnetic resonancespectrometry and scanning electron microscopy. Theoretical calculations usingthe MM+ HyperChem force field showed a preference for inclusion of thesidechain of the epibrassinolide molecule into the -cyclodextrin cavity toform a 1:1 inclusion complex, although complexes involving inclusion ofthe steroidal nucleus also possess a favourable interaction energy. Rice laminainclination assay, employing IAC-103 and IAC-104 cultivars, showed an improvedactivity for the epibrassinolide-cyclodextrin complex compared to theepibrassinolide itself. The results suggest that brassinosteroid complexationwith cyclodextrins may enhance the biological activity of these plant growthregulators.     

Improvement of the lytic properties of a β-1,3-glucanase by directed evolution

BGLII is a bacterial endoglucanase that hydrolyzes the β-1,3-glucan present in yeast cell walls, resulting in lysis of Saccharomyces cerevisiae. As a result of this property, BGLII is considered a potential tool for downstream processing and recovery of biotechnological products produced in yeast. Here we describe the improvement of the yeast lytic activity of BGLII, achieved by a directed evolution approach involving random mutagenesis and screening for variants with improved catalytic activity, combined with site-directed mutagenesis. A BGLII variant having three times the wild-type hydrolytic activity on laminarin was identified. The purified enzyme also exhibited higher lytic activity on yeast cells. Mutations causing the improvements are located very close to each other in the amino acid sequence, suggesting that the region should be considered as a target for further improvements of the glucanase activity. These results demonstrate the feasibility of molecular evolution methods for the improvement of the BGLII hydrolytic activity, and open a window for further improvement of this or other properties in glycosyl hydrolases in general.     

The β-1,3-glucan-binding protein from the crayfish Pacifastacus leniusculus,when reacted with a β-1,3-glucan,induces spreading and degranulation of crayfish granular cells

Summary A -1,3-glucan-binding protein (GBP) was purified from crayfish plasma, and incubated with laminarin (L), a -1,3-glucan. The GBP reacted with laminarin (GBP-L) induced strong spreading and partial degranulation of isolated and separated crayfish granular haemocytes. However, neither the GBP nor laminarin alone induced any changes in the crayfish granular cells. When monolayers of granular haemocytes were incubated with 20 g of GBP-L, more than 82% of the haemocytes were affected. The activity of GBP-L on granular cells was dose-dependent and a plateau was reached at 10 g of GBP-L. The degranulation of crayfish haemocytes induced by GBP-L seemed to occur by a regulated exocytosis, since it was strongly inhibited by specific blockers of this process such as SITS or calmidazolium. Monospecific anti-GBP antibodies also totally blocked the effect of GBP-L on crayfish granular cells. Indirect immunofluoresence staining demonstrated that the GBP-L could bind to the surface of granular cells, whereas GBP did not bind or bound very weakly to the haemocyte surface.     

Purification and Partial Characterization of a Novel β-1,3-Endoglucanase from Streptomyces rutgersensis

A β-1,3-endoglucanase produced by Streptomyces rutgersensis was purified to a homogeneity by the fractional precipitation with ammonium sulfate, ion exchange chromatography on Q-Sepharose and hydrophobic chromatography on Butyl Sepharose. A typical procedure provided 11.74-fold purification with 12.53 % yield. SDS-PAGE of the purified protein showed one protein band. The exact molecular mass of the enzyme obtained by mass spectrometry was 41.25 kDa; the isoelectric point was between pH 4.2–4.4. The optimal β-glucanase catalytic activity was at pH 7 and 50 °C. An enzyme was only active toward glucose polymers containing β-1,3 linkages and hydrolyzed Saccharomyces cerevisiae cell wall β-glucan in an endo-like way: reaction products were different molecular size β-glucans, which were larger than glucose.     

Study on the interaction between the inclusion complex of hematoxylin with β-cyclodextrin and DNA

Ultraviolet-visible (UV-vis) spectra, fluorescence spectra, electrochemistry, and the thermodynamic method were used to discuss the interaction mode between the inclusion complex of hematoxylin with β-cyclodextrin and herring sperm DNA. On the condition of physiological pH, the result showed that hematoxylin and β-cyclodextrin formed an inclusion complex with binding ratio n(hematoxylin):n(β-cyclodextrin) = 1:1. The interaction mode between β-cyclodextrin-hematoxylin and DNA was a mixed binding, which contained intercalation and electrostatic mode. The binding ratio between β-cyclodextrin-hematoxylin and DNA was n(β-cyclodextrin -hematoxylin):n(DNA) = 2:1, binding constant was K(?)(298.15K) = 5.29 × 10? L·mol?1, and entropy worked as driven force in this action.     

Modulation of aggregation of silk fibroin by synergistic effect of the complex of curcumin and β-cyclodextrin

Amyloid aggregation has been associated with numerous human pathological diseases. A recent study has demonstrated that silk fibroin intermittently endorses amyloidogenesis in vivo. In the current study, we explored the propensity of silk fibroin to undergo amyloid-like aggregation and its prevention using an optimized concoction of curcumin with β-cyclodextrin. Aggregation of silk fibroin resulted in the formation of fibrils with a diameter of ~3.2 nm. However, addition of the optimized concentration of curcumin and β-cyclodextrin to silk fibroin inhibited aggregation and preserved the random coil conformation even under aggregation inducing conditions, as demonstrated by CD and FTIR spectroscopy. Benzene rings of curcumin interact with the aromatic residues of fibroin via hydrophobic interactions. However, β-cyclodextrin preferentially interacts with the non-polar residues, which are the core components for nucleation dependent protein aggregation. The present study demonstrates the ability of the concoction of curcumin and β-cyclodextrin in tuning the self assembly process of fibroin. It also provides a platform to explore the assembly process of nano-fibril and hierarchical structures in vitro along with a novel insight for designing clinically relevant silk-based functional biomaterials.     

Novel M4 positive allosteric modulators derived from questioning the role and impact of a presumed intramolecular hydrogen-bonding motif in β-amino carboxamide-harboring ligands

This letter describes a focused exercise to explore the role of the β-amino carboxamide moiety found in all of the first generation M₄ PAMs and question if the NH₂ group served solely to stabilize an intramolecular hydrogen bond (IMHB) and enforce planarity. To address this issue (and to potentially find a substitute for the β-amino carboxamide that engendered P-gp and contributed to solubility liabilities), we removed the NH₂, generating des-amino congeners and surveyed other functional groups in the β-position. These modifications led to weak M₄ PAMs with poor DMPK properties. Cyclization of the β-amino carboxamide moiety by virtue of a pyrazole ring re-enforced the IMHB, led to potent (and patented) M₄ PAMs, many as potent as the classical bicyclic β-amino carboxamide analogs, but with significant CYP1A2 inhibition. Overall, this exercise indicated that the β-amino carboxamide moiety most likely facilitates an IMHB, and is essential for M₄ PAM activity within classical bicyclic M₄ PAM scaffolds.     

Ligand-induced formation of a transient tryptophan synthase complex with αββ subunit stoichiometry

The prototypical tryptophan synthases form a stable heterotetrameric αββα complex in which the constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and outside (sTrpB2a) the tryptophan operon. It has previously been shown that sTrpB2a does not functionally or structurally interact with sTrpA, whereas sTrpB2i substantially activates sTrpA in a unidirectional manner. However, in the absence of catalysis, no physical complex between sTrpB2i and sTrpA could be detected. In order to elucidate the structural requirements for complex formation, we have analyzed the interaction between sTrpA (α-monomer) and sTrpB2i (ββ-dimer) by means of spectroscopy, analytical gel filtration, and analytical ultracentrifugation, as well as isothermal titration calorimetry. In the presence of the TrpA ligand glycerol 3-phosphate (GP) and the TrpB substrate l-serine, sTrpA and sTrpB2i formed a physical complex with a thermodynamic dissociation constant of about 1 μM, indicating that the affinity between the α- and ββ-subunits is weaker by at least 1 order of magnitude than the affinity between the corresponding subunits of prototypical tryptophan synthases. The observed stoichiometry of the complex was 1 subunit of sTrpA per 2 subunits of sTrpB2i, which corresponds to a αββ quaternary structure and testifies to a strong negative cooperativity for the binding of the α-monomers to the ββ-dimer. The analysis of the interaction between sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that the αββ complex remains stable during the whole catalytic cycle and disintegrates into α- and ββ-subunits upon the release of the reaction product tryptophan. The formation of a transient tryptophan synthase complex, together with the observed low affinity of sTrpB2i for l-serine, couples the rate of tryptophan biosynthesis in S. solfataricus to the cytosolic availability of l-serine.     

Relationship between pellet formation by Aspergillus oryzae strain KB and the production of β-fructofuranosidase with high transfructosylation activity

Aspergillus oryzae KB produces two β-fructofuranosidases (F1 and F2). F1 has high transfructosylation activity (Ut) to produce fructooligosaccharides. F2 has high hydrolysis activity (Uh), releasing glucose and fructose. It is desirable to selectively produce F1, which can be used for production of fructooligosaccharides. Here, the relationship between filamentous pellet size and selective production of F1 in liquid culture was investigated. Our finding revealed that: (i) The mean particle size of pellets (5.88 ± 1.36 mm) was larger, and the ratio of Ut to Uh was improved (Ut/Uh = 5.0) in 10% sucrose medium compared with 1% sucrose medium (pellet size = 2.60 ± 0.37 mm; Ut/Uh = 0.96). (ii) The final culture pH of the 1% sucrose medium was 8.7; on controlling the pH of 1% sucrose medium at 5.0, increased pellet size (9.69 ± 2.01 mm) and Ut/Uh (7.8) were observed. (iii) When 3% glycerin was used as carbon source, the pellet size decreased to 1.09 ± 0.33 mm and Ut/Uh was 0.57. (iv) In medium containing 1% sucrose, the pellet size was dependent on the number of spores used in the culture inoculum, but, in these experiments, Ut/Uh was almost constant (1.05 ± 0.08). Collectively, the data show that the value of Ut/Uh is proportional to the pellet size when liquid culture of A. oryzae strain KB is performed in some conditions (such as in the presence of high sucrose concentration, low pH, or added Tween surfactant), but in other conditions Ut/Uh is independent of pellet size.     

Fibril formation from the amyloid-β peptide is governed by a dynamic equilibrium involving association and dissociation of the monomer

Here I review the molecular mechanisms by which water-soluble monomeric amyloid-β (Aβ) peptides are transformed into well-organized supramolecular complexes called amyloid fibrils. The mechanism of amyloid formation is considered theoretically on the basis of experimental results, and the structural and mechanistic similarities of amyloid fibrils to three-dimensional crystals are highlighted. A number of important results from the literature are described. These include the observation that a correct ratio of monomer association and dissociation rate constants is key for formation of well-organized amyloid fibrils. The dynamic nature of the amyloid-β structure is discussed, along with the possibly obligate requirement of the transient formation of a hairpin-like fold prior to its incorporation into amyloid fibrils. Many rounds of monomer association and dissociation events may be present during an apparently silent lag-period. Amongst these association/dissociation events, interaction between the C-terminal regions of the Aβ peptide seems to be more favored. Such association and dissociation events occurring in a “trial-and-error” fashion may be an important requirement for the formation of well-organized amyloid fibrils.     

Biochemical Characterization of a β-1,3-Glucanase from Trichoderma koningii Induced by Cell Wall of Rhizoctonia solani

Trichoderma species are readily isolated from Brazilian cerrado soil by conventional methods and some of them were characterized as Trichoderma koningii. The effect of carbon source on the production of β-1,3-glucanases in the culture filtrates of a specific Trichoderma koningii strain (ALL 13) was investigated. Enzyme activity was detected in all carbon sources tested and only one band of β-1,3-glucanase was detected in non-denaturing PAGE. This enzyme was purified by Sephacryl S-200 gel filtration and Phenyl Sepharose CL 4B chromatography. A typical procedure provided 105-fold purification with 13.4% yield. The molecular weight of the purified enzyme was 75 kDa as estimated by SDS-PAGE. The enzyme hydrolyzed laminarin in an endo-like fashion to form small oligosaccharides and glucose. The K_m and V_max values for β-1,3-glucanase, using laminarin as substrate, were 0.148 mg.mL⁻¹ and 0.159 U.min⁻¹, respectively. The pH optimum for the enzyme was pH 4.6 and maximum activity was obtained at 50°C. Hg²⁺ inhibited the purified enzyme.     

Effect of complex formation by taxifolin and naringenin with Cu(i) ions on the distribution of the components of complexes in the octanol–water system

The interaction of two structurally close flavanones: taxifolin and naringenin with copper(I) ions and its effect on the distribution of flavonoids and the corresponding ions in a biphasic system octanol–water have been studied. It has been shown that these polyphenols form complexes with copper ions of different stoichiometric ratio depending on the pH of medium (5.4, 7.4, and 9.0). The interaction of the flavonoids with copper ions leads to an increase in the fraction of polyphenols in the water phase at all pH values examined. The fraction of metal ions in octanol in the presence of both taxifolin and naringenin is maximal in the range of neutral pH values. The parameters obtained in the study, such as the partition coefficient and the coefficient of distribution in a biphasic system octanol–water (logP and logD) form the physicochemical basis necessary for the estimation of the bioavailability of flavonoids and the corresponding metal ions upon their combined consumption.     

Crystal structures of the laminarinase catalytic domain from Thermotoga maritima MSB8 in complex with inhibitors: essential residues for β-1,3- and β-1,4-glucan selection

Laminarinases hydrolyzing the β-1,3-linkage of glucans play essential roles in microbial saccharide degradation. Here we report the crystal structures at 1.65-1.82 ? resolution of the catalytic domain of laminarinase from the thermophile Thermotoga maritima with various space groups in the ligand-free form or in the presence of inhibitors gluconolactone and cetyltrimethylammonium. Ligands were bound at the cleft of the active site near an enclosure formed by Trp-232 and a flexible GASIG loop. A closed configuration at the active site cleft was observed in some molecules. The loop flexibility in the enzyme may contribute to the regulation of endo- or exo-activity of the enzyme and a preference to release laminaritrioses in long chain carbohydrate hydrolysis. Glu-137 and Glu-132 are proposed to serve as the proton donor and nucleophile, respectively, in the retaining catalysis of hydrolyzation. Calcium ions in the crystallization media are found to accelerate crystal growth. Comparison of laminarinase and endoglucanase structures revealed the subtle difference of key residues in the active site for the selection of β-1,3-glucan and β-1,4-glucan substrates, respectively. Arg-85 may be pivotal to β-1,3-glucan substrate selection. The similarity of the structures between the laminarinase catalytic domain and its carbohydrate-binding modules may have evolutionary relevance because of the similarities in their folds.     

Membrane-bound structure and alignment of the antimicrobial β-sheet peptide gramicidin S derived from angular and distance constraints by solid state 19F-NMR

The antimicrobial properties of the cyclic -sheet peptide gramicidin S are attributed to its destabilizing effect on lipid membranes. Here we present the membrane-bound structure and alignment of a derivative of this peptide, based on angular and distance constraints. Solid-state ¹⁹F-NMR was used to study a ¹⁹F-labelled gramicidin S analogue in dimyristoylphosphatidylcholine bilayers at a lipid:peptide ratio of 80:1 and above. Two equivalent leucine side chains were replaced by the non-natural amino acid 4F-phenylglycine, which serves as a highly sensitive reporter on the structure and dynamics of the peptide backbone. Using a modified CPMG multipulse sequence, the distance between the two ¹⁹F-labels was measured from their homonuclear dipolar coupling as 6 Å, in good agreement with the known backbone structure of natural gramicidin S in solution. By analyzing the anisotropic chemical shift of the ¹⁹F-labels in macroscopically oriented membrane samples, we determined the alignment of the peptide in the bilayer and described its temperature-dependent mobility. In the gel phase, the ¹⁹F-labelled gramicidin S is aligned symmetrically with respect to the membrane normal, i.e., with its cyclic -sheet backbone lying flat in the plane of the bilayer, which is fully consistent with its amphiphilic character. Upon raising the temperature to the liquid crystalline state, a considerable narrowing of the ¹⁹F-NMR chemical shift dispersion is observed, which is attributed the onset of global rotation of the peptide and further wobbling motions. This study demonstrates the potential of the ¹⁹F nucleus to describe suitably labelled polypeptides in membranes, requiring only little material and short NMR acquisition times.     

Purification,characterization and sequencing of the major β-1,3-glucanase from the midgut of Tenebrio molitor larvae

The major β-1,3-glucanase from Tenebrio molitor (TLam) was purified to homogeneity (yield, 6%; enrichment, 113 fold; specific activity, 4.4 U/mg). TLam has a molecular weight of 50 kDa and a pH optimum of 6. It is an endoglucanase that hydrolyzes β-1,3-glucans as laminarin and yeast β-1,3-1,6-glucan, but is inactive toward other polysaccharides (as unbranched β-1,3-glucans or mixed β-1,3-1,4-glucan from cereals) or disaccharides. The enzyme is not inhibited by high substrate concentrations and has low processivity (0.6). TLam has two ionizable groups involved in catalysis, and His, Tyr and Arg residues plus a divalent ion at the active site. A Cys residue important for TLam activity is exposed after laminarin binding. The cDNA coding for this enzyme was cloned and sequenced. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLam activity. TLam efficiently lyses fungal cells, suggesting a role in making available walls and cell contents to digestion and in protecting the midgut from pathogen infections.     

Flower formation in the short-day plant Kalanchoë by grafting with a long-day and a short-long-day Echeveria

Flower formation was induced in the shortday plant Kalanchoë blossfeldiana Poellnitz under long-day conditions by grafting with flowering shoots of the short-long-day plant Echeveria harmsii (Rose) MacBr. and of the long-day plant Echeveria pulvoliver (E. pulvinata Rose x E. harmsii). Vegetative shoots from induced Echeveria plants failed to cause a flowering response in Kalanchoë. The presence of flowering and vegetative shoots side by side on induced Echeveria plants provides evidence for physiological chimeras in this genus.Abbreviations LD long day(s) - SD short day(s) - LDP long-day plant(s) - LSDP long-short-day plant(s) - SDP short-day plant(s) - SLDP short-long-day plant(s)