Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Molecular and active-site structure of a Bacillus 1,3-1,4-beta-glucanase.

ABSTRACT: The three-dimensional structure of the hybrid Bacillus 1,3-1,4-beta-glucanase (beta-glucanase; 1,3-1,4-beta-D-glucan 4-glucanohydrolase, lichenase, EC 3.2.1.73) designated H(A16-M) was determined by x-ray crystallography at a resolution of 2.0 A and refined to an R value of 16.4% using stereochemical restraints. The protein molecule consists mainly of two seven-stranded antiparallel beta-pleated sheets arranged atop each other to form a compact, sandwich-like structure. A channel crossing one side of the protein molecule accommodates an inhibitor, 3,4-epoxybutyl beta-D-cellobioside, which binds covalently to the side chain of Glu-105, as seen in a crystal structure analysis at 2.8-A resolution of the protein-inhibitor complex (R = 16.8%). That Glu-105 may be indispensible for enzyme catalysis by H(A16-M) is suggested by site-directed mutagenesis of this residue, which inevitably leads to an inactive enzyme.

SUBMITTER: Keitel T

PROVIDER: S-EPMC46701 | biostudies-other | 1993 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Molecular and active-site structure of a Bacillus 1,3-1,4-beta-glucanase.

Keitel T T Simon O O Borriss R R Heinemann U U

Proceedings of the National Academy of Sciences of the United States of America 19930601 11

The three-dimensional structure of the hybrid Bacillus 1,3-1,4-beta-glucanase (beta-glucanase; 1,3-1,4-beta-D-glucan 4-glucanohydrolase, lichenase, EC 3.2.1.73) designated H(A16-M) was determined by x-ray crystallography at a resolution of 2.0 A and refined to an R value of 16.4% using stereochemical restraints. The protein molecule consists mainly of two seven-stranded antiparallel beta-pleated sheets arranged atop each other to form a compact, sandwich-like structure. A channel crossing one si ...[more]

PMID: 8099449

Similar Datasets

Pre-steady-state kinetics of Bacillus licheniformis 1,3-1,4-beta-glucanase: evidence for a regulatory binding site.

Project description:In a previous paper, we reported the first stopped-flow experiments on a Bacillus licheniformis 1,3-1,4-beta-glucanase [Abel, Planas and Christensen (2001) Biochem. J. 357, 195-202]. It was shown that the pre-steady-state kinetics of the 1,3-1,4-beta-glucanase using the substrate 4-methylumbelliferyl 3-O-beta-cellobiosyl-beta-D-glucoside may be explained by a reaction scheme involving an induced fit and the binding of two substrates as well as a second enzymic conformational change, whereas the results definitely could not be explained in terms of the simple double-displacement scheme. In the present study, we report further stopped-flow kinetic results on the glucanase using a series of low-molecular-mass substrates with various leaving groups and varying chain length. The analysis of the resulting data leads to the conclusion that the free enzyme exists in two conformations, one of which binds the substrates rather strongly in a regulatory site, before any productive interactions can take place. This corresponds to an allosteric activation mechanism. With these substrates, however, the productive enzyme-substrate species are also able to change into less active or inactive forms. This may be seen as a feedback inhibitory mechanism.

| S-EPMC1223346 | biostudies-other

Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis.

Project description:1,3-1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C) were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm) compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.

| S-EPMC4839689 | biostudies-literature

Beta-1,4-glucanase-like protein from the cyanobacterium Synechocystis PCC6803 is a beta-1,3-1,4-glucanase and functions in salt stress tolerance.

Project description:In the present study, we characterized the gene (Cyanobase accession number slr0897) designated Ssglc encoding a beta-1,4-glucanase-like protein (SsGlc) from Synechocystis PCC6803. The deduced amino acid sequence for Ssglc showed a high degree of similarity to sequences of GH (glycoside hydrolase) family 9 beta-1,4-glucanases (cellulases) from various sources. Surprisingly, the recombinant protein obtained from the Escherichia coli expression system was able to hydrolyse barley beta-glucan and lichenan (beta-1,3-1,4-glucan), but not cellulose (beta-1,4-glucan), curdlan (beta-1,3-glucan), or laminarin (beta-1,3-1,6-glucan). A 1H-NMR analysis of the enzymatic products revealed that the enzyme hydrolyses the beta-1,4-glycosidic linkage of barley beta-glucan through an inverting mechanism. The data indicated that SsGlc was a novel type of GH9 glucanase which could specifically hydrolyse the beta-1,3-1,4-linkage of glucan. The growth of mutant Synechocystis cells in which the Ssglc gene was disrupted by a kanamycin-resistance cartridge gene was almost the same as that of the wild-type cells under continuous light (40 micromol of photons/m2 per s), a 12 h light (40 micromol of photons/m2 per s)/12 h dark cycle, cold stress (4 degrees C), and high light stress (200 micromol of photons/m2 per s). However, under salt stress (300-450 mM NaCl), growth of the Ssglc-disrupted mutant cells was significantly inhibited as compared with that of the wild-type cells. The Ssglc-disrupted mutant cells showed a decreased rate of O2 consumption and NaHCO3-dependent O2 evolution as compared with the wild-type cells under salt stress. Under osmotic stress (100-400 mM sorbitol), there was no difference in growth between the wild-type and the Ssglc-disrupted mutant cells. These results suggest that SsGlc functions in salt stress tolerance in Synechocystis PCC6803.

| S-EPMC1925248 | biostudies-literature

Action of an endo-?-1,3(4)-glucanase on cellobiosyl unit structure in barley ?-1,3:1,4-glucan.

Project description:?-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single ?-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-?-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley ?-1,3:1,4-glucan. To find the minor structure on which the endo-?-1,3(4)-glucanase acts, we prepared oligosaccharides from barley ?-1,3:1,4-glucan by endo-?-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-?-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as ?-Glc-1,3-?-Glc-1,4-?-Glc-1,3-?-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-?-1,3(4)-glucanase acts on the cellobiosyl units of barley ?-1,3:1,4-glucan in an endo-manner.

| S-EPMC4673573 | biostudies-literature

Improved barley broiler feed with transgenic malt containing heat-stable (1,3-1,4)-beta-glucanase.

Project description:The low nutritional value of barley for poultry is because of the absence of an intestinal enzyme for efficient depolymerization of (1, 3-1,4)-beta-glucan, the major polysaccharide of the endosperm cell walls. This leads to high viscosity in the intestine, limited nutrient uptake, decreased growth rate, and unhygienic sticky droppings adhering to chickens and floors of the production cages. Consequently, the 7.5 billion broiler chickens produced annually in the United States are primarily raised on corn-soybean diets. Here we show that addition to normal barley of 6.2% transgenic malt containing a thermotolerant (1,3-1,4)-beta-glucanase (4.28 microg.g(-1) soluble protein) provides a weight gain equivalent to corn diets. The number of birds with adhering sticky droppings is drastically reduced. Intestines and excrements of chickens fed the barley control diet contained large amounts of soluble (1,3-1,4)-beta-glucan, which was reduced by 75 and 50%, respectively, by adding transgenic malt to the diet. The amount of active recombinant enzyme in the small intestine corresponded to that present in the feed, whereas an 11-fold concentration of the enzyme was observed in the ceca, and a 7.5-fold concentration occurred in the excrement. Glycosylation of the beta-glucanase isolated from the ceca testified to its origin from the transgenic barley. Analysis of the data from this trial demonstrates the possibility of introducing individual recombinant enzymes into various parts of the gastrointestinal tract of chickens with transgenic malt and thereby the possibility of evaluating their effect on the metabolism of a given ingredient targeted by the enzyme.

| S-EPMC17606 | biostudies-literature

Preliminary X-ray diffraction analysis of a thermophilic β-1,3-1,4-glucanase from Clostridium thermocellum.

Project description:β-1,3-1,4-Glucanases catalyze the specific hydrolysis of internal β-1,4-glycosidic bonds adjacent to the 3-O-substituted glucose residues in mixed-linked β-glucans. The thermophilic glycoside hydrolase CtGlu16A from Clostridium thermocellum exhibits superior thermal profiles, high specific activity and broad pH adaptability. Here, the catalytic domain of CtGlu16A was expressed in Escherichia coli, purified and crystallized in the trigonal space group P3121, with unit-cell parameters a=b=74.5, c=182.9 Å, by the sitting-drop vapour-diffusion method and diffracted to 1.95 Å resolution. The crystal contains two protein molecules in an asymmetric unit. Further structural determination and refinement are in progress.

| S-EPMC4089538 | biostudies-literature

Presteady-state kinetics of Bacillus 1,3-1,4-beta-glucanase: binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate.

Project description:In the present study the first stopped-flow experiments performed on Bacillus 1,3-1,4-beta-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-beta-cellobiosyl-beta-D-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100 s(-1), was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme-substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.

| S-EPMC1221941 | biostudies-other

Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase.

Project description:Two genes, xynD and gluB, encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase (lichenase) from Bacillus polymyxa have been cloned and expressed in Escherichia coli and Bacillus subtilis. A sequenced DNA fragment of 4,466 bp contains both genes, which are separated by 155 bp. The xynD and gluB genes encode proteins of 67.8 kDa (XYND) and 27 kDa (GLUB). Two peptides with molecular masses of 62 and 53 kDa appear in cell extracts of E. coli and culture supernatants of B. subtilis clones containing the xynD gene. Both peptides show xylanase activity in zymogram analysis. The XYND enzyme also shows alpha-L-arabinofuranosidase activity. The XYND peptide and the xylanase XYNZ from Clostridium thermocellum (O. Grépinet, M. C. Chebrou, and P. Béguin, J. Bacteriol. 170:4582-4588, 1988) show 64% homology in a stretch of about 280 amino acids.

| S-EPMC212542 | biostudies-other

Cloning and DNA sequencing of bgaA, a gene encoding an endo-beta-1,3-1,4-glucanase, from an alkalophilic Bacillus strain (N137).

Project description:The gene bgaA encoding an alkaline endo-beta-1,3-1,4-glucanase (lichenase) from an alkalophilic Bacillus sp. strain N137, isolated in our laboratory, was cloned and expressed from its own promoter in Escherichia coli. The nucleotide sequence of a 1,416-bp DNA fragment containing bgaA was determined and revealed an open reading frame of 828 nucleotides. The deduced protein sequence consists of 276 amino acids and has a 31-amino-acid putative signal peptide which is functional in E. coli, in which the BgaA protein is located mainly in the periplasmic space. The lichenase activity of BgaA is stable between pH 6 and 12, it shows optimal activity at a temperature between 60 and 70 degrees C, and it retains 65% of its activity after incubation at 70 degrees C for 1 h. This protein is similar to some other lichenases from Bacillus species such as B. amyloliquefaciens, B. brevis, B. licheniformis, B. macerans, B. polymyxa, and B. subtilis. However, it has a lysine-rich region at the carboxy terminus which is not found in any other published lichenase sequence and might be implicated in the unusual biochemical properties of this enzyme. The location of the mRNA 5' end was determined by primer extension and corresponds to nucleotide 235. A typical Bacillus sigma A promoter precedes the transcription start site.

| S-EPMC201461 | biostudies-other

Overexpression of an acidic endo-β-1,3-1,4-glucanase in transgenic maize seed for direct utilization in animal feed.

Project description:BackgroundIncorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the anti-nutritional factors, for efficient nutrition absorption. The acidic endo-β-1,3-1,4-glucanase (Bgl7A) from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed.Methodology/principal findingsHere we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A) by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The β-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The β-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0-8.0, which is in agreement with the normal environment of digestive tract.Conclusion/significanceOur study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.

| S-EPMC3876984 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data