Structural analysis of rice Os4BGlu18 monolignol ß-glucosidase.

Monolignol glucosides are storage forms of monolignols, which are polymerized to lignin to strengthen plant cell walls. The conversion of monolignol glucosides to monolignols is catalyzed by monolignol ß-glucosidases. Rice Os4BGlu18 ß-glucosidase catalyzes hydrolysis of the monolignol glucosides, coniferin, syringin, and p-coumaryl alcohol glucoside more efficiently than other natural substrates. To understand more clearly the basis for substrate specificity of a monolignol ß-glucosidase, the structure of Os4BGlu18 was determined by X-ray crystallography. Crystals of Os4BGlu18 and its complex with δ-gluconolactone diffracted to 1.7 and 2.1 Å resolution, respectively. Two protein molecules were found in the asymmetric unit of the P212121 space group of their isomorphous crystals. The Os4BGlu18 structure exhibited the typical (ß/α)8 TIM barrel of glycoside hydrolase family 1 (GH1), but the four variable loops and two disulfide bonds appeared significantly different from other known structures of GH1 ß-glucosidases. Molecular docking studies of the Os4BGlu18 structure with monolignol substrate ligands placed the glycone in a similar position to the δ-gluconolactone in the complex structure and revealed the interactions between protein and ligands. Molecular docking, multiple sequence alignment, and homology modeling identified amino acid residues at the aglycone-binding site involved in substrate specificity for monolignol ß-glucosides. Thus, the structural basis of substrate recognition and hydrolysis by monolignol ß-glucosidases was elucidated.

Identification of Phenolic Compounds from Nettle as New Candidate Inhibitors of Main Enzymes Responsible on Type-II Diabetes.

BACKGROUND: In medicinal chemistry, the discovery of small organic molecules that can be optimized and lead to a future drug capable of effectively modulating the biological activity of a therapeutic target remains a major challenge. Because of the harmful secondary effects of synthesized therapeutic molecules, the development of research has been oriented towards phytomedicines. Phenolic compounds from medicinal plants are constantly explored for new therapeutic use. METHODS: In this paper, we studied interactions between main enzymes responsible for causing type 2 diabetes mellitus (T2DM) and phenolic compounds from nettle (Urtica dioica L.) using molecular Docking with Molecular Operating Environment Software (MOE). RESULTS: Docking results show a common molecule (secoisolariciresinol), which may form stable complexes with depeptidyl peptidase 4 (DPP-4), alpha-amylase and beta-glucosidase with binding energy of -7.04732084 kcal/mol, -3.82946181 kcal/mol and -4.16077089 kcal/mol respectively. Besides secoisolariciresinol, other phenolic compounds give better docking score than the original co-crystallized ligand for alpha-amylase (PDB ID 5U3A) and beta-glucosidase (PDB ID 1OGS). CONCLUSION: The obtained results are promising for the discovery of new alpha-amylase and betaglucosidase inhibitors. This study also confirms the folk use of nettle as antidiabetic agent.

Rapid mechanochemical encapsulation of biocatalysts into robust metal-organic frameworks.

Metal-organic frameworks (MOFs) have recently garnered consideration as an attractive solid substrate because the highly tunable MOF framework can not only serve as an inert host but also enhance the selectivity, stability, and/or activity of the enzymes. Herein, we demonstrate the advantages of using a mechanochemical strategy to encapsulate enzymes into robust MOFs. A range of enzymes, namely ß-glucosidase, invertase, ß-galactosidase, and catalase, are encapsulated in ZIF-8, UiO-66-NH2, or Zn-MOF-74 via a ball milling process. The solid-state mechanochemical strategy is rapid and minimizes the use of organic solvents and strong acids during synthesis, allowing the encapsulation of enzymes into three prototypical robust MOFs while maintaining enzymatic biological activity. The activity of encapsulated enzyme is demonstrated and shows increased resistance to proteases, even under acidic conditions. This work represents a step toward the creation of a suite of biomolecule-in-MOF composites for application in a variety of industrial processes.

Functional and structural characterization of a ß-glucosidase involved in saponin metabolism from intestinal bacteria.

Saponins are natural glycosides widely used in medicine and the food industry. Although saponin metabolism in human is dependent on intestinal microbes, few involving bacteria enzymes have been identified. We cloned BlBG3, a GH3 ß-glucosidase from Bifidobacterium longum, from human stool. We found that BlBG3 catalyzes the hydrolysis of glycoside furostanol and ginsenoside Rb1 at higher efficiency than other microbial ß-glucosidases. Structural analysis of BlBG3 in complex with d-glucose revealed its three unique loops, which form a deep pocket and participate in substrate binding. To understand how substrate is bound to the pocket, molecular docking was performed and the binding interactions of protobioside with BlBG3 were revealed. Mutational study suggested that R484 and H642 are critical for enzymatic activity. Our study presents the first structural and functional analysis of a saponin-processing enzyme from human microbiota.

Production and distribution of beta-glucosidase in a mutant strain Trichoderma viride T 100-14.

The characterization of beta-glucosidase's production and distribution in a mutant strain Trichoderma viride T 100-14 at extracellular and intracellular levels were studied in this paper. Three experiment groups were done automatically with pH controlled at 4.8 during fermentation process, with 1mg/ml 2-deoxy-d-glucose addition or without pH control and 2-deoxy-d-glucose addition (control). Activity assay and electron microscopic immunogold labeling experiments were performed at different culture periods (24, 48, 72, 96 and 120 hours). Under constant pH 4.8, high density of immunogold labeling particles, highest intracellular enzyme activity, total enzyme activity and specific activity were observed at 24 hours of fermentation. After 72 hours, the extracellular and total activities fluctuated little and the maximal activity in extracellular fraction was 2.7 times higher than control. By contrast, with 2-deoxy-d-glucose addition, the secreted and total beta-glucosidase activities achieved their maximum at 96 hours of fermentation, and the maximal secreted activity increased 2.05-fold than the control. Additionally, the secretion ratio (maximal secreted beta-glucosidase activity/maximal total activity) with pH control or 2-deoxy-d-glucose addition was elevated profoundly near to a level as the cellulase in fungi.

Formation of fibrillar multimers of oat beta-glucosidase isoenzymes is mediated by the As-Glu1 monomer.

Oat beta-glucosidase (EC 3.2.1.21) exists in two isomeric forms of homomultimer (type I) and heteromultimer (type II), which are comprised of two 60 kDa monomers of As-Glu1 and As-Glu2. The cDNA of As-Glu2 was cloned in this study, whereas As-Glu1 was previously cloned as As-P60. The As-Glu2 cDNA encodes a plastid-directing transit peptide of 57 amino acid residues and a mature protein of 521 amino acid residues. The amino acid sequence of As-Glu2 is highly homologous to that of As-Glu1, except for their C-terminal portions. When the two cDNAs of the mature proteins were expressed as T7.Tag-fused proteins in Escherichia coli, they produced soluble and enzymatically active T7.Tag-As-Glu1 and T7.Tag-As-Glu2 proteins. The T7.Tag-As-Glu1 was assembled into a donut-shaped hexamer ring which was in turn stacked in integer numbers to form long fibrillar homomultimers of different lengths with a molecular mass of up to several million daltons. On the other hand, the T7.Tag-As-Glu2 primarily formed a dimer rather than a multimer. When both cDNAs of As-Glu1 and As-Glu2 were co-expressed as T7.Tag-fused mature proteins, they were also assembled into a hexamer ring comprised of the two monomers in a 1:1 stoichiometry. The heteromeric hexamer was stacked in smaller numbers to form the heteromultimer of T7. Tag-As-Glu1 and -As-Glu2. The results indicate that the As-Glu1 monomer plays a crucial role in the formation of both the As-Glu1 homomultimer and the As-Glu1 and As-Glu2 heteromultimer. We describe here a unique structure for the oat beta-glucosidase fibrillar multimer that is formed by stacking the hexamer rings composed of As-Glu1 and/or As-Glu2.

Structure of the N-terminal cellulose-binding domain of Cellulomonas fimi CenC determined by nuclear magnetic resonance spectroscopy.

Multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine the tertiary structure of the 152 amino acid N-terminal cellulose-binding domain from Cellulomonas fimi 1,4-beta-glucanase CenC (CBDN1). CBDN1 was studied in the presence of saturating concentrations of cellotetraose, but due to spectral overlap, the oligosaccharide was not included in the structure calculations. A total of 1705 interproton nuclear Overhauser effect (NOE), 56 phi, 88 psi, 42 chi 1, 9 chi 2 dihedral angle, and 88 hydrogen-bond restraints were used to calculate 25 final structures. These structures have a rmsd from the average of 0.79 +/- 0.11 A for all backbone atoms excluding disordered termini and 0.44 +/- 0.05 A for residues with regular secondary structures. CBDN1 is composed of 10 beta-strands, folded into two antiparallel beta-sheets with the topology of a jelly-roll beta-sandwich. The strands forming the face of the protein previously determined by chemical shift perturbations to be responsible for cellooligosaccharide binding [Johnson, P. E., Tomme, P., Joshi, M. D., & McIntosh, L. P. (1996) Biochemistry 35, 13895-13906] are shorter than those forming the opposite side of the protein. This results in a 5-stranded binding cleft, containing a central strip of hydrophobic residues that is flanked on both sides by polar hydrogen-bonding groups. The presence of this cleft provides a structural explanation for the unique selectivity of CBDN1 for amorphous cellulose and other soluble oligosaccharides and the lack of binding to crystalline cellulose. The tertiary structure of CBDN1 is strikingly similar to that of the bacterial 1,3-1,4-beta-glucanases, as well as other sugar-binding proteins with jelly-roll folds.

Immunocytochemistry of plant defense mechanisms induced upon microbial attack.

During the past few years, cyto- and immunocytochemical techniques have been developed and widely used for locating and identifying various molecules in plant cell compartments. The last decade has witnessed tremendous improvements in molecular cytology, thus allowing an accurate in situ detection of various components thought to play important biological functions in the plant metabolism. The use of immunocytochemistry to investigate resistance mechanisms of plants upon pathogen attack has provided key information on the defense strategy that plants elaborate during a host-pathogen interaction. Of the various proteins induced in response to infection, chitinases and beta-1,3-glucanases have been the focus of particular attention due to their believed antimicrobial activity through the hydrolysis of the main fungal wall components, chitin and beta-1,3-glucans. Attention has also been paid to beta-fructosidase, the enzyme that hydrolyzes sucrose into glucose and fructoside. The marked accumulation of this enzyme upon pathogen infection has led to the consideration that infection may greatly influence the metabolic activity of colonized tissues by creating alterations of source-sink relationships. Another facet of the plant's defense strategy that has been the focus of considerable interest is related to the accumulation of structural compounds, such as hydroxyproline-rich glycoproteins and callose, to reinforce the wall architecture, thus decreasing vulnerability to microbial enzymes. A number of alternatives designed to improve plant protection towards pathogen invasion have been suggested. Among these, the production of transgenic plants expressing constitutively a foreign resistance gene and the pretreatment of plants with elicitors of defense reactions have been the subject of intensive studies at the molecular, biochemical, and cytological levels. Results of such studies clearly demonstrate the important contribution that cyto- and immunocytochemical approaches can make to our knowledge of how plants defend themselves and how plant disease resistance can be directly enhanced. These approaches will undoubtedly be active areas for future research in the development of biological control alternatives in which the mode of action of the product used is of key importance.