The Experts below are selected from a list of 528 Experts worldwide ranked by ideXlab platform

Anastassios C Papageorgiou - One of the best experts on this subject based on the ideXlab platform.

  • crystal structure of a cu zn superoxide dismutase from the thermophilic fungus Chaetomium thermophilum
    Protein and Peptide Letters, 2021
    Co-Authors: Imran Mohsin, Liqing Zhang, Anastassios C Papageorgiou
    Abstract:

    Background Thermophilic fungi have recently emerged as a promising source of thermostable enzymes. Superoxide dismutases are key antioxidant metalloenzymes with promising therapeutic effects in various diseases, both acute and chronic. However, structural heterogeneity and low thermostability limit their therapeutic efficacy. Objective Although several studies from hypethermophilic superoxide dismutases (SODs) have been reported, information about Cu,Zn-SODs from thermophilic fungi is scarce. Chaetomium thermophilum is a thermophilic fungus that could provide proteins with thermophilic properties. Method The enzyme was expressed in Pichia pastoris cells and crystallized using the vapor-diffusion method. X-ray data were collected, and the structure was determined and refined to 1.56 A resolution. Structural analysis and comparisons were carried out. Results The presence of 8 molecules (A through H) in the asymmetric unit resulted in four different interfaces. Molecules A and F form the typical homodimer which is also found in other Cu,Zn-SODs. Zinc was present in all subunits of the structure while copper was found in only four subunits with reduced occupancy (C, D, E and F). Conclusion The ability of the enzyme to form oligomers and the elevated Thr:Ser ratio may be contributing factors to its thermal stability. Two hydrophobic residues that participate in interface formation and are not present in other CuZn-SODs may play a role in the formation of new interfaces and the oligomerization process. The CtSOD crystal structure reported here is the first Cu,Zn-SOD structure from a thermophilic fungus.

  • crystal structure of a gh3 β glucosidase from the thermophilic fungus Chaetomium thermophilum
    International Journal of Molecular Sciences, 2019
    Co-Authors: Imran Mohsin, Nirmal Poudel, Anastassios C Papageorgiou
    Abstract:

    Beta-glucosidases (β-glucosidases) have attracted considerable attention in recent years for use in various biotechnological applications. They are also essential enzymes for lignocellulose degradation in biofuel production. However, cost-effective biomass conversion requires the use of highly efficient enzymes. Thus, the search for new enzymes as better alternatives of the currently available enzyme preparations is highly important. Thermophilic fungi are nowadays considered as a promising source of enzymes with improved stability. Here, the crystal structure of a family GH3 β-glucosidase from the thermophilic fungus Chaetomium thermophilum (CtBGL) was determined at a resolution of 2.99 A. The structure showed the three-domain architecture found in other β-glucosidases with variations in loops and linker regions. The active site catalytic residues in CtBGL were identified as Asp287 (nucleophile) and Glu517 (acid/base). Structural comparison of CtBGL with Protein Data Bank (PDB)-deposited structures revealed variations among glycosylated Asn residues. The enzyme displayed moderate glycosylation compared to other GH3 family β-glucosidases with similar structure. A new glycosylation site at position Asn504 was identified in CtBGL. Moreover, comparison with respect to several thermostability parameters suggested that glycosylation and charged residues involved in electrostatic interactions may contribute to the stability of the enzyme at elevated temperatures. The reported CtBGL structure provides additional insights into the family GH3 enzymes and could offer new ideas for further improvements in β-glucosidases for more efficient use in biotechnological applications regarding cellulose degradation.

  • crystal structure and biological implications of a glycoside hydrolase family 55 β 1 3 glucanase from Chaetomium thermophilum
    Biochimica et Biophysica Acta, 2017
    Co-Authors: Anastassios C Papageorgiou, Jinyin Chen
    Abstract:

    Crystal structures of a β-1,3-glucanase from the thermophilic fungus Chaetomium thermophilum were determined at 1.20 and 1.42A resolution in the free and glucose-bound form, respectively. This is the third structure of a family 55 glycoside hydrolase (GH55) member and the second from a fungus. Based on comparative structural studies and site-directed mutagenesis, Glu654 is proposed as the catalytic acid residue. The substrate binding cleft exhibits restricted access on one side, rendering the enzyme as an exo-β-1,3-glucanase as confirmed also by thin layer chromatography experiments. A lack of stacking interactions was found at the substrate binding cleft, suggesting that interactions at positions -1, +1 and +2 are sufficient to orientate the substrate. A binding pocket was identified that could explain binding of branched laminarin and accumulation of laminaritriose.

  • expression purification and crystallization of a family 55 β 1 3 glucanase from Chaetomium thermophilum
    Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2015
    Co-Authors: Anastassios C Papageorgiou
    Abstract:

    A β-1,3-glucanase from the thermophilic fungus Chaetomium thermophilum was overexpressed in Pichia pastoris, purified and crystallized in the presence of 1.8 M sodium/potassium phosphate pH 6.8 as a precipitant. Data to 2.0 A resolution were collected in-house at 293 K from a single crystal. The crystal was found to belong to space group P2(1), with unit-cell parameters a = 64.1, b = 85.8, c = 68.5 A, β = 93.1° and one molecule in the asymmetric unit.

  • crystal structure and biochemical characterization of a manganese superoxide dismutase from Chaetomium thermophilum
    Biochimica et Biophysica Acta, 2014
    Co-Authors: Teemu Haikarainen, Anastassios C Papageorgiou, Clemence Frioux, Liqing Zhnag
    Abstract:

    A manganese superoxide dismutase from the thermophilic fungus Chaetomium thermophilum (CtMnSOD) was expressed in Pichia pastoris and purified to homogeneity. Its optimal temperature was 60°C with approximately 75% of its activity retained after incubation at 70°C for 60min. Recombinant yeast cells carrying C. thermophilum mnsod gene exhibited higher stress resistance to salt and oxidative stress-inducing agents than control yeast cells. In an effort to provide structural insights, CtMnSOD was crystallized and its structure was determined at 2.0A resolution. The overall architecture of CtMnSOD was found similar to other MnSODs with highest structural similarities obtained against a MnSOD from the thermotolerant fungus Aspergillus fumigatus. In order to explain its thermostability, structural and sequence analysis of CtMnSOD with other MnSODs was carried out. An increased number of charged residues and an increase in the number of intersubunit salt bridges and the Thr:Ser ratio were identified as potential reasons for the thermostability of CtMnSOD.

Ed Hurt - One of the best experts on this subject based on the ideXlab platform.

  • global transcriptome characterization and assembly of the thermophilic ascomycete Chaetomium thermophilum
    Genes, 2021
    Co-Authors: A Singh, Ed Hurt, Nikola Kellner, Géza Schermann, Sven Reislohner, Michael Brunner
    Abstract:

    A correct genome annotation is fundamental for research in the field of molecular and structural biology. The annotation of the reference genome of Chaetomium thermophilum has been reported previously, but it is essentially limited to open reading frames (ORFs) of protein coding genes and contains only a few noncoding transcripts. In this study, we identified and annotated full-length transcripts of C. thermophilum by deep RNA sequencing. We annotated 7044 coding genes and 4567 noncoding genes. Astonishingly, 23% of the coding genes are alternatively spliced. We identified 679 novel coding genes as well as 2878 novel noncoding genes and corrected the structural organization of more than 50% of the previously annotated genes. Furthermore, we substantially extended the Gene Ontology (GO) and Enzyme Commission (EC) lists, which provide comprehensive search tools for potential industrial applications and basic research. The identified novel transcripts and improved annotation will help to understand the gene regulatory landscape in C. thermophilum. The analysis pipeline developed here can be used to build transcriptome assemblies and identify coding and noncoding RNAs of other species.

  • global transcriptome characterization and assembly of thermophilic ascomycete Chaetomium thermophilum
    bioRxiv, 2019
    Co-Authors: A Singh, Ed Hurt, Nikola Kellner, Géza Schermann, S Reisloehner, Michael Brunner
    Abstract:

    A correct genome annotation is fundamental for research in the field of molecular and structural biology. The annotation of the reference genome Chaetomium thermophilum has been reported previously, but it is limited to open reading frames (ORFs) of genes and contains only a few noncoding transcripts. In this study, we identified and annotated by deep RNA sequencing full-length transcripts of C.thermophilum. We annotated 7044 coding genes and a large number of noncoding genes (n=4567). Astonishingly, 23% of the coding genes are alternatively spliced. We identified 679 novel coding genes and corrected the structural organization of more than 50% of the previously annotated genes. Furthermore, we substantially extended the Gene Ontology (GO) and Enzyme Commission (EC) lists, which provide comprehensive search tools for potential industrial applications and basic research. The identified novel transcripts and improved annotation will help understanding the gene regulatory landscape in C.thermophilum. The analysis pipeline developed here can be used to build transcriptome assemblies and identify coding and noncoding RNAs of other species. The R packages for gene and GO annotation database can be found under https://www.bzh.uni-heidelberg.de/brunner/Chaetomium_thermophilum.

  • The UTP-A complex from Chaetomium thermophilum.
    2017
    Co-Authors: Fabiola R. Calviño, Ed Hurt, Markus Kornprobst, Géza Schermann, Fabienne Birkle, Klemens Wild, Tamas Fischer, Yasar Luqman Ahmed, Irmgard Sinning
    Abstract:

    (A) Scheme showing the spatial assembly of the fungal UTP-A complex including the Utp4 X-ray structure and the EM-modeled 5'-ETS. The propellers of remaining UTP-A proteins (Utp8, Utp15, and 2× Utp17) are placed according previous biochemical and EM-studies. The α-solenoidal parts (including whole Utp5) are not included. The entire Utp10 molecule turning around Utp4 is interpreted as also the very C-terminus (atomic model) of Utp8 next to the Velcro-closure of Utp4. The position of the disease-modified arginine in human Utp4 in the interface to Utp10 is highlighted within a red sphere. (B) Comparison of the UTP-A complexes from Chaetomium thermophilum (left panel; [9]) and Saccharomyces cerevisiae (right panel; [27, 28]). While the overall architecture is conserved, the 5'-end of the RNA shows a different arrangement. In addition, the Upt8-Utp4 contact is not visible in the yeast structures.

  • In vitro protein-RNA UV crosslinking analysis of ctUtp4.
    2017
    Co-Authors: Fabiola R. Calviño, Ed Hurt, Markus Kornprobst, Géza Schermann, Fabienne Birkle, Klemens Wild, Tamas Fischer, Yasar Luqman Ahmed, Irmgard Sinning
    Abstract:

    (A) Hits obtained from deep sequencing analysis of Chaetomium thermophilum His6-Utp4 (coverage, blue) mapped within the 5'-ETS (nucleotides 1 to 587) after UTP-A/5'-ETS RNP assembly by co-expression in yeast. (B) Mutations (deletions and substitutions) identified after cDNA library synthesis are indicated by red bars. Mutational hot spots observed in the two crosslinked regions are labeled accordingly (G66 and A220). As background control, the UTP-A/5'-ETS complex carrying untagged Utp4 (“no His6 tag”) was used. The crosslinked region around 5'-ETS bases 100–140, which was found also in the untagged control, is marked with an asterisk. (C) and (D) The two main regions of the 5'-ETS (A53-C96 and A192-C235) that were crosslinked to His6-Utp4 are shown together the number of mutations per base. The respective 5'-ETS sequence is depicted below. Mutational hot spots G66 and A220 colored in red.

  • developing genetic tools to exploit Chaetomium thermophilum for biochemical analyses of eukaryotic macromolecular assemblies
    Scientific Reports, 2016
    Co-Authors: Nikola Kellner, Johannes Schwarz, Miriam Sturm, Javier Fernandezmartinez, Sabine Griesel, Wenzhu Zhang, Brian T Chait, Michael P Rout, Ulrich Kuck, Ed Hurt
    Abstract:

    We describe a method to genetically manipulate Chaetomium thermophilum, a eukaryotic thermophile, along with various biochemical applications. The transformation method depends on a thermostable endogenous selection marker operating at high temperatures combined with chromosomal integration of target genes. Our technique allows exploiting eukaryotic thermophiles as source for purifying thermostable native macromolecular complexes with an emphasis on the nuclear pore complex, holding great potential for applications in basic science and biotechnology.

Daniel Kummel - One of the best experts on this subject based on the ideXlab platform.

  • architecture and mechanism of the late endosomal rab7 like ypt7 guanine nucleotide exchange factor complex mon1 ccz1
    Nature Communications, 2017
    Co-Authors: Stephan Kiontke, Lars Langemeyer, Anne Kuhlee, Saskia Schuback, Stefan Raunser, Christian Ungermann, Daniel Kummel
    Abstract:

    The Mon1-Ccz1 complex (MC1) is the guanine nucleotide exchange factor (GEF) for the Rab GTPase Ypt7/Rab7 and is required for endosomal maturation and fusion at the vacuole/lysosome. Here we present the overall architecture of MC1 from Chaetomium thermophilum, and in combining biochemical studies and mutational analysis in yeast, we identify the domains required for catalytic activity, complex assembly and localization of MC1. The crystal structure of a catalytic MC1 core complex bound to Ypt7 provides mechanistic insight into its function. We pinpoint the determinants that allow for a discrimination of the Rab7-like Ypt7 over the Rab5-like Vps21, which are both located on the same membrane. MC1 shares structural similarities with the TRAPP complex, but employs a novel mechanism to promote nucleotide exchange that utilizes a conserved lysine residue of Ypt7, which is inserted upon MC1 binding into the nucleotide-binding pocket of Ypt7 and contributes to specificity.

Santiago Ramonmaiques - One of the best experts on this subject based on the ideXlab platform.

  • structural insight into the core of cad the multifunctional protein leading de novo pyrimidine biosynthesis
    Structure, 2017
    Co-Authors: Maria Morenomorcillo, A Grandegarcia, Alba Ruizramos, Francisco Del Canoochoa, Jasminka Boskovic, Santiago Ramonmaiques
    Abstract:

    Summary CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines in animals, self-assembles into ∼1.5 MDa hexamers. The structures of the dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains of human CAD have been previously determined, but we lack information on how these domains associate and interact with the rest of CAD forming a multienzymatic unit. Here, we prove that a construct covering human DHO and ATC oligomerizes as a dimer of trimers and that this arrangement is conserved in CAD-like from fungi, which holds an inactive DHO-like domain. The crystal structures of the ATC trimer and DHO-like dimer from the fungus Chaetomium thermophilum confirm the similarity with the human CAD homologs. These results demonstrate that, despite being inactive, the fungal DHO-like domain has a conserved structural function. We propose a model that sets the DHO and ATC complex as the central element in the architecture of CAD.

Joao Atilio Jorge - One of the best experts on this subject based on the ideXlab platform.

  • a novel α glucosidase from Chaetomium thermophilum var coprophilum that converts maltose into trehalose purification and partial characterisation of the enzyme
    Process Biochemistry, 2006
    Co-Authors: Giovana Cristina Giannesi, Maria De Lourdes Teixeira De Moraes Polizeli, Hector Francisco Terenzi, Joao Atilio Jorge
    Abstract:

    Abstract A constitutive extracellular α-glucosidase from Chaetomium thermophilum var. coprophilum was purified and characterised. The enzyme exhibited a carbohydrate content of 14% and an apparent molecular mass of 107 kDa, estimated by gel filtration. Analysis by SDS-PAGE revealed two polypeptide bands of 22 and 26 kDa, suggesting that the enzyme was constituted of two subunits of 22 kDa and two subunits of 26 kDa. Optima of pH and temperature were 7.0 and 60 °C, respectively. The enzyme hydrolysed maltose, amylose, starch, maltooligosaccharides and isomaltose, in order of preference. The enzyme exhibited transglycosylation activity producing trehalose and maltooligosaccharides from 5% maltose. At early stages of the transglycosylation reaction trehalose was the predominant product; at later stages maltooligosaccharides were also accumulated. Trehalose inhibited both glycosyl hydrolase and transglycosylase activities. To our knowledge this is the first report of a α-glucosidase from a filamentous fungus that in addition to glycosyl hydrolase activity produces trehalose by transglycosylation.

  • extracellular β d glucosidase from Chaetomium thermophilum var coprophilum production purification and some biochemical properties
    Journal of Basic Microbiology, 2002
    Co-Authors: Leandra Lorice Venturi, Maria De Lourdes Teixeira De Moraes Polizeli, Hector Francisco Terenzi, Rosa Dos Prazeres Melo Furriel, Joao Atilio Jorge
    Abstract:

    The thermophilic fungus Chaetomium thermophilum var. coprophilum produced large amounts of extracellular and intracellular beta-glucosidase activity when grown on cellulose or cellobiose as carbon sources. The presence of glucose in the culture medium drastically decreased the level of beta-glucosidase activity, while cycloheximide prevented the induction of the extracellular enzyme activity by cellobiose. An extracellular beta-glucosidase induced by avicel was purified by a procedure involving acetone precipitation and chromatography on two DEAE-cellulose columns. The purified enzyme was a basic protein, with a carbohydrate content of 73%. The deglycosylated enzyme exhibited a molecular mass of 43 kDa, with pH and temperature optima of 5.5 and 65 degrees C respectively. The beta-glucosidase hydrolysed only cellobiose and p-nitrophenyl-beta-D-glucopyranoside, exhibiting apparent Km values of 3.13 mM and 0.76 mM, respectively. The native purified enzyme was stable up to 2 hours at 60 degrees C, and its thermal stability was directly dependent on glycosylation.

  • biochemical characterization of a ca2 dependent acid trehalase activity from the thermophilic fungus Chaetomium thermophilum var coprophilum
    Fems Microbiology Letters, 1999
    Co-Authors: Elzira Maria De Almeida, Hector Francisco Terenzi, Maria De Lourdes Polizeli, Joao Atilio Jorge
    Abstract:

    A trehalase activity was purified from the mycelium of Chaetomium thermophilum var. corpophilum. The enzyme was composed of two identical polypeptides of apparent molecular mass of 98 kDa. The pI of the enzyme was about 3.9. The purified trehalase was stimulated by calcium, manganese and cobalt and inhibited by EDTA, ADP and ATP. The enzyme exhibited a Km of 0.63 mM, optimum pH of 6.5, and optimum temperature of 55°C. The C. thermophilum trehalase is another example of calcium-dependent, thermophilic acid trehalases which exhibit mixed properties of regulated (neutral) and non-regulated (acid) trehalases.