scholarly journals Calcium-induced tertiary structure modifications of endo-β-1,3-glucanase from Pyrococcus furiosus in 7.9 M guanidinium chloride

2005 ◽  
Vol 386 (3) ◽  
pp. 515-524 ◽  
Author(s):  
Roberta CHIARALUCE ◽  
Giulio GIANESE ◽  
Sebastiana ANGELACCIO ◽  
Rita FLORIO ◽  
Johan F. T. van LIESHOUT ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Pyrococcus Furiosus ◽  
Glycoside Hydrolases ◽  
Guanidinium Chloride ◽  
Absorption Region ◽  
Native Form ◽  
Carboxylate Groups ◽  
The Family ◽  
Notable Increase ◽  
Putative Binding Site

The family 16 endo-β-1,3 glucanase from the extremophilic archaeon Pyrococcus furiosus is a laminarinase, which in 7.9 M GdmCl (guanidinium chloride) maintains a significant amount of tertiary structure without any change of secondary structure. The addition of calcium to the enzyme in 7.9 M GdmCl causes significant changes to the near-UV CD and fluorescence spectra, suggesting a notable increase in the tertiary structure which leads to a state comparable, but not identical, to the native state. The capability to interact with calcium in 7.9 M GdmCl with a consistent recovery of native tertiary structure is a unique property of this extremely stable endo-β-1,3 glucanase. The effect of calcium on the thermodynamic parameters relative to the GdmCl-induced equilibrium unfolding has been analysed by CD and fluorescence spectroscopy. The interaction of calcium with the native form of the enzyme is studied by Fourier-transform infrared spectroscopy in the absorption region of carboxylate groups and by titration in the presence of a chromophoric chelator. A homology-based model of the enzyme is generated and used to predict the putative binding site(s) for calcium and the structural interactions potentially responsible for the unusual stability of this protein, in comparison with other family 16 glycoside hydrolases.

scholarly journals Tertiary structure in 7.9 m guanidinium chloride − the role of Glu53 and Asp287 in Pyrococcus furiosus endo-β-1,3-glucanase

FEBS Journal ◽  
2007 ◽  
Vol 274 (23) ◽  
pp. 6167-6179 ◽  
Author(s):  
Roberta Chiaraluce ◽  
Rita Florio ◽  
Sebastiana Angelaccio ◽  
Giulio Gianese ◽  
Johan F. T. van Lieshout ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Pyrococcus Furiosus ◽  
Guanidinium Chloride

Tertiary structure in 7.9 m guanidinium chloride − the role of Glu53 and Asp287 in Pyrococcus furiosus endo-β-1,3-glucanase

FEBS Journal ◽  
2007 ◽  
Vol 0 (0) ◽  
pp. 071115064009001-???
Author(s):  
Roberta Chiaraluce ◽  
Rita Florio ◽  
Sebastiana Angelaccio ◽  
Giulio Gianese ◽  
Johan F. T. van Lieshout ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Pyrococcus Furiosus ◽  
Guanidinium Chloride

Positive selection of three chitinase genes of the family 18 of glycoside hydrolases in mammals

Biologia ◽  
2009 ◽  
Vol 64 (4) ◽  
Author(s):  
Beibei He ◽  
Li Wang ◽  
Jinhong Wang ◽  
Gang Li ◽  
Shuyi Zhang
Keyword(s):  
Positive Selection ◽  
Catalytic Domain ◽  
Digestive Enzyme ◽  
Glycoside Hydrolases ◽  
Wide Range ◽  
The Family ◽  
Maximum Likelihood Methods ◽  
Mammalian Genomes ◽  
Acidic Mammalian Chitinase ◽  
Chitinase Genes

AbstractThe digestive enzyme chitinase degrades chitin, and is found in a wide range of organisms, from prokaryotes to eukaryotes. Although mammals cannot synthesize or assimilate chitin, several proteins of the glycoside hydrolase (GH) chitinase family GH18, including some with enzymatic activity, have recently been identified from mammalian genomes. Consequently, there is growing interest in molecular evolution of this family of proteins. Here we report on the use of maximum likelihood methods to test for evidence of positive selection in three genes of the chitinase family GH18, all of which are found in mammals. These focal genes are CHIA, CHIT1 and CHI3L1, which encode the chitinase proteins acidic mammalian chitinase, chitotriosidase and cartilage protein 39, respectively. The results of our analyses indicate that each of these genes has undergone independent selective pressure in their evolution. Additionally, we have found evidence of a signature of positive natural selection, with most sites identified as being subject to adaptive evolution located in the catalytic domain. Our results suggest that positive selection on these genes stems from their function in digestion and/or immunity.

scholarly journals Improved Thermostability of Clostridium thermocellum Endoglucanase Cel8A by Using Consensus-Guided Mutagenesis

2012 ◽  
Vol 78 (9) ◽  
pp. 3458-3464 ◽  
Author(s):  
Michael Anbar ◽  
Ozgur Gul ◽  
Raphael Lamed ◽  
Ugur O. Sezerman ◽  
Edward A. Bayer
Keyword(s):  
Sequence Alignment ◽  
Clostridium Thermocellum ◽  
Structural Changes ◽  
Glycoside Hydrolases ◽  
Wild Type ◽  
Triple Mutant ◽  
Content Type ◽  
The Family ◽  
Catalytic Module ◽  
Glycine Substitution

ABSTRACTThe use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, fromClostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem2:997–1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that ofC. thermocellumCel8A.

scholarly journals Financial products used by South African households at different life stages

2017 ◽  
Vol 9 (1) ◽  
pp. 310-325
Author(s):  
Cinzia Stedall ◽  
Jan M.P. Venter
Keyword(s):  
South African ◽  
Financial Literacy ◽  
Household Head ◽  
Life Stages ◽  
Product Usage ◽  
Single Parent Families ◽  
Financial Products ◽  
The Family ◽  
Notable Increase ◽  
Selection Of

South African households don’t save enough to ensure financial freedom after retirement. This article poses the following question: do life stages have a significant impact on the financial products used by households? The question is asked in order to identify possible interventions that could increase financial freedom. This study found that life stages have a significant impact on South African households’ selection of financial products. The use of financial products for each of the levels of the financial product usage hierarchy increases as the age of the household head increases and when the size of the family increases, the only exception being single-parent families. The study indicated that financial literacy programmes should focus on young couples and young families, as there is a notable increase in their financial product usage. The study also found a very low usage of wealth management products by South African households and suggests that policymakers consider the introduction of an incentive to increase household’s usage of these products.

scholarly journals A Novel Virus Discovered in the Yeast Pichia membranifaciens.

2022 ◽  
Author(s):  
Mark D Lee ◽  
Jack W Creagh ◽  
Lance R Fredericks ◽  
Angela M Crabtree ◽  
Jagsish Suresh Patel ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Yeast Species ◽  
Molecular Models ◽  
Viral Origin ◽  
Gag Protein ◽  
Pichia Membranifaciens ◽  
The Family ◽  
Dsrna Viruses ◽  
Novel Virus

Mycoviruses are widely distributed across fungi, including yeasts of the Saccharomycotina subphylum. It was recently discovered that the yeast species Pichia membranifaciens contained double stranded RNAs (dsRNAs) that were predicted to be of viral origin. The fully sequenced dsRNA is 4,578 bp in length, with RNA secondary structures similar to the packaging, replication, and frameshift signals of totiviruses of the family Totiviridae. This novel virus has been named Pichia membranifaciens virus L-A (PmV-L-A) and is related to other totiviruses previously described within the Saccharomycotina yeasts. PmV-L-A is part of a monophyletic subgroup within the I-A totiviruses, implying a common ancestry between mycoviruses isolated from the Pichiaceae and Saccharomycetaceae yeasts. Energy minimized AlphaFold2 molecular models of the PmV-L-A Gag protein revealed structural conservation with the previously solved structure of the Saccharomyces cerevisiae virus L-A (ScV-L-A) Gag protein. The predicted tertiary structure of the PmV-L-A Pol and its homologs provide details of the potential mechanism of totivirus RNA-dependent RNA polymerases (RdRps) because of structural similarities to the RdRps of mammalian dsRNA viruses. Insights into the structure, function, and evolution of totiviruses gained from yeasts is important because of their parallels with mammalian viruses and the emerging role of totiviruses in animal disease.

scholarly journals Action of Designer Cellulosomes on HomogeneousVersusComplex Substrates

2005 ◽  
Vol 280 (16) ◽  
pp. 16325-16334 ◽  
Author(s):  
Henri-Pierre Fierobe ◽  
Florence Mingardon ◽  
Adva Mechaly ◽  
Anne Bélaïch ◽  
Marco T. Rincon ◽  
...  
Keyword(s):  
Self Assembly ◽  
The Self ◽  
Glycoside Hydrolases ◽  
Synergistic Activity ◽  
Multiprotein Complex ◽  
And Topology ◽  
Processive Endoglucanase ◽  
The Family ◽  
Major Factors ◽  
Cellulose Binding

In recent work (Fierobe, H.-P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belaïch, A., Lamed, R., Shoham, Y., and Belaich, J.-P. (2002)J. Biol. Chem. 277, 49621–49630), we reported the self-assembly of a comprehensive set of defined “bifunctional” chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.

scholarly journals Crystallization and preliminary X-ray diffraction data of β-galactosidase fromAspergillus niger

2014 ◽  
Vol 70 (11) ◽  
pp. 1529-1531 ◽  
Author(s):  
Agustín Rico-Díaz ◽  
Ángel Vizoso Vázquez ◽  
M. Esperanza Cerdán ◽  
Manuel Becerra ◽  
Julia Sanz-Aparicio
Keyword(s):  
Diffraction Data ◽  
Metal Ion ◽  
Hydrolytic Activity ◽  
Glycoside Hydrolases ◽  
X Ray Diffraction ◽  
Data Set ◽  
X Ray ◽  
The Family ◽  
Crystallization Experiments ◽  
Metal Ion Affinity Chromatography

β-Galactosidase fromAspergillus niger(An-β-Gal), belonging to the family 35 glycoside hydrolases, hydrolyzes the β-galactosidase linkages in lactose and other galactosides. It is extensively used in industry owing to its high hydrolytic activity and safety. The enzyme has been expressed in yeasts and purified by immobilized metal-ion affinity chromatography for crystallization experiments. The recombinant An-β-Gal, deglycosylated to avoid heterogeneity of the sample, has a molecular mass of 109 kDa. Rod-shaped crystals grew using PEG 3350 as the main precipitant agent. A diffraction data set was collected to 1.8 Å resolution.

scholarly journals A novel β-xylosidase structure fromGeobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

2014 ◽  
Vol 70 (5) ◽  
pp. 1366-1374 ◽  
Author(s):  
Giannina Espina ◽  
Kirstin Eley ◽  
Guillaume Pompidor ◽  
Thomas R. Schneider ◽  
Susan J. Crennell ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Glycoside Hydrolase ◽  
Thermophilic Bacterium ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Gene Encoding ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Family ◽  
Hydrolase Family

Geobacillus thermoglucosidasiusis a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding aG. thermoglucosidasiusβ-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed inEscherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme–substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of theG. thermoglucosidasiusβ-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.

scholarly journals Molecular Characterization of Novel Family IV and VIII Esterases from a Compost Metagenomic Library

2021 ◽  
Vol 9 (8) ◽  
pp. 1614
Author(s):  
Jong-Eun Park ◽  
Geum-Seok Jeong ◽  
Hyun-Woo Lee ◽  
Hoon Kim
Keyword(s):  
Metagenomic Library ◽  
Industrial Applications ◽  
Native Form ◽  
Metagenome Library ◽  
The Family ◽  
Alkaline Conditions ◽  
Molecular Masses ◽  
Novel Esterase ◽  
Family Iv

Two novel esterase genes, est8L and est13L, were isolated and identified from a compost metagenomic library. The encoded Est8L and Est13L had molecular masses of 33,181 and 44,913 Da consisting of 314 and 411 amino acids, respectively, without signal peptides. Est8L showed the highest identity (32.9%) to a hyper-thermophilic carboxylesterase AFEST from Archaeoglobus fulgidus compared to other esterases reported and was classified to be a novel member of family IV esterases with conserved regions such as HGGG, DY, GXSXG, DPL, and GXIH. Est13L showed the highest identity (98.5%) to the family VIII esterase Est7K from the metagenome library. Est8L and Est13L had the highest activities for p-nitrophenyl butyrate (C4) and p-nitrophenyl caproate (C6), respectively, and Est13L showed a broad substrate specificity for p-nitrophenyl substrates. Est8L and Est13L effectively hydrolyzed glyceryl tributyrate. The optimum temperatures for activities of Est8L and Est13L were identical (40 °C), and the optimum pH values were 9.0 and 10.0, respectively. Est13L showed higher thermostability than Est8L. Sephacryl S-200 HR chromatography showed that the native form of Est8L was a dimer. Interestingly, Est13L was found to be a tetramer, contrary to other family VIII esterases reported. Est8L was inhibited by 30% isopropanol, methanol, and acetonitrile; however, Est13L was activated to 182.9% and 356.1%, respectively, by 30% isopropanol and methanol. Est8L showed enantioselectivity for the S-form, but Est13L showed no enantioselectivity. These results show that intracellular Est8L and/or Est13L are oligomeric in terms of native forms and can be used for pharmaceutical and industrial applications with organic solvents under alkaline conditions.

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