Thermophilic Fungus Chaetomium Thermophilum Research Articles

Expression, purification, and biochemical analysis of the RNA-DNA hybrid helicase Sen1/SETX from Chaetomium thermophilum.

Yeast Sen1 and its vertebrate ortholog Senataxin (also known as SETX) are RNA-DNA resolving helicases. Sen1 and SETX are implicated in multiple critical nuclear functions not limited to but including DNA replication and repair, RNA processing, and transcription. These>200kDa helicases have a two-domain architecture with an N-terminal regulatory helical repeat array linked to an SF1b helicase motor core via a variable sized central linker of low complexity sequence. Given the size of these proteins, production of milligram quantities of protein that is suitable for biochemical, biophysical, and protein structural analysis has been challenging. To overcome these limitations, we developed a robust selectable high-yield YFP-fusion protein expression method for Sen1 production in mammalian cells, followed by purification on a high-affinity YFP-binding camelid nanobody support. Herein, we detail methods and protocols for the expression and purification of recombinant Sen1 from the thermophilic fungus Chaetomium thermophilum, and the quantitative characterization of its RNA-DNA duplex resolution activity.

Identification and characterization of sugar-regulated promoters in Chaetomium thermophilum

The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5’ untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.

Conformational changes in mitochondrial complex I of the thermophilic eukaryote Chaetomium thermophilum.

Mitochondrial complex I is a redox-driven proton pump that generates proton-motive force across the inner mitochondrial membrane, powering oxidative phosphorylation and ATP synthesis in eukaryotes. We report the structure of complex I from the thermophilic fungus Chaetomium thermophilum, determined by cryoEM up to 2.4-Å resolution. We show that the complex undergoes a transition between two conformations, which we refer to as state 1 and state 2. The conformational switch is manifest in a twisting movement of the peripheral arm relative to the membrane arm, but most notably in substantial rearrangements of the Q-binding cavity and the E-channel, resulting in a continuous aqueous passage from the E-channel to subunit ND5 at the far end of the membrane arm. The conformational changes in the complex interior resemble those reported for mammalian complex I, suggesting a highly conserved, universal mechanism of coupling electron transport to proton pumping.

The iron–sulfur cluster assembly (ISC) protein Iba57 executes a tetrahydrofolate-independent function in mitochondrial [4Fe–4S] protein maturation

Mitochondria harbor the bacteria-inherited iron–sulfur cluster assembly (ISC) machinery to generate [2Fe–2S; iron–sulfur (Fe–S)] and [4Fe–4S] proteins. In yeast, assembly of [4Fe–4S] proteins specifically involves the ISC proteins Isa1, Isa2, Iba57, Bol3, and Nfu1. Functional defects in their human equivalents cause the multiple mitochondrial dysfunction syndromes, severe disorders with a broad clinical spectrum. The bacterial Iba57 ancestor YgfZ was described to require tetrahydrofolate (THF) for its function in the maturation of selected [4Fe–4S] proteins. Both YgfZ and Iba57 are structurally related to an enzyme family catalyzing THF-dependent one-carbon transfer reactions including GcvT of the glycine cleavage system. On this basis, a universally conserved folate requirement in ISC-dependent [4Fe–4S] protein biogenesis was proposed. To test this idea for mitochondrial Iba57, we performed genetic and biochemical studies in Saccharomyces cerevisiae, and we solved the crystal structure of Iba57 from the thermophilic fungus Chaetomium thermophilum. We provide three lines of evidence for the THF independence of the Iba57-catalyzed [4Fe–4S] protein assembly pathway. First, yeast mutants lacking folate show no defect in mitochondrial [4Fe–4S] protein maturation. Second, the 3D structure of Iba57 lacks many of the side-chain contacts to THF as defined in GcvT, and the THF-binding pocket is constricted. Third, mutations in conserved Iba57 residues that are essential for THF-dependent catalysis in GcvT do not impair Iba57 function in vivo, in contrast to an exchange of the invariant, surface-exposed cysteine residue. We conclude that mitochondrial Iba57, despite structural similarities to both YgfZ and THF-binding proteins, does not utilize folate for its function.

Non-canonical binding of the Chaetomium thermophilum PolD4 N-terminal PIP motif to PCNA involves Q-pocket and compact 2-fork plug interactions but no 310 helix.

DNA polymerase δ (Pol δ) is a key enzyme for the maintenance of genome integrity in eukaryotic cells, acting in concert with the sliding clamp processivity factor PCNA (proliferating cell nuclear antigen). Three of the four subunits of human Pol δ interact directly with the PCNA homotrimer via a short, conserved protein sequence known as a PCNA interacting protein (PIP) motif. Here, we describe the identification of a PIP motif located towards the N terminus of the PolD4 subunit of Pol δ (equivalent to human p12) from the thermophilic filamentous fungus Chaetomium thermophilum and present the X-ray crystal structure of the corresponding peptide bound to PCNA at 2.45 Å. Like human p12, the fungal PolD4 PIP motif displays non-canonical binding to PCNA. However, the structures of the human p12 and fungal PolD4 PIP motif peptides are quite distinct, with the fungal PolD4 PIP motif lacking the 310 helical segment that characterises most previously identified PIP motifs. Instead, the fungal PolD4 PIP motif binds PCNA via conserved glutamine that inserts into the Q-pocket on the surface of PCNA and with conserved leucine and phenylalanine sidechains forming a compact 2-fork plug that inserts into the hydrophobic pocket on PCNA. Despite the unusual binding mode of the fungal PolD4, isothermal calorimetry (ITC) measurements show that its affinity for PCNA is similar to that of its human orthologue. These observations add to a growing body of information on how diverse proteins interact with PCNA and highlight how binding modes can vary significantly between orthologous PCNA partner proteins.

Coupling proteomics and metabolomics for the unsupervised identification of protein-metabolite interactions in Chaetomium thermophilum.

Protein–metabolite interactions play an important role in the cell’s metabolism and many methods have been developed to screen them in vitro. However, few methods can be applied at a large scale and not alter biological state. Here we describe a proteometabolomic approach, using chromatography to generate cell fractions which are then analyzed with mass spectrometry for both protein and metabolite identification. Integrating the proteomic and metabolomic analyses makes it possible to identify protein-bound metabolites. Applying the concept to the thermophilic fungus Chaetomium thermophilum, we predict 461 likely protein-metabolite interactions, most of them novel. As a proof of principle, we experimentally validate a predicted interaction between the ribosome and isopentenyl adenine.

Novel Proteome and N-Glycoproteome of the Thermophilic Fungus Chaetomium thermophilum in Response to High Temperature.

Thermophilic fungi are eukaryotic species that grow at high temperatures, but little is known about the underlying basis of thermophily at cell and molecular levels. Here the proteome and N-glycoproteome of Chaetomium thermophilum at varying culture temperatures (30, 50, and 55°C) were studied using hydrophilic interaction liquid chromatography enrichment and high-resolution liquid chromatography–tandem mass spectroscopy analysis. With respect to the proteome, the numbers of differentially expressed proteins were 1,274, 1,374, and 1,063 in T50/T30, T55/T30, and T55/T50, respectively. The upregulated proteins were involved in biological processes, such as protein folding and carbohydrate metabolism. Most downregulated proteins were involved in molecular functions, including structural constituents of the ribosome and other protein complexes. For the N-glycoproteome, the numbers of differentially expressed N-glycoproteins were 160, 176, and 128 in T50/T30, T55/T30, and T55/T50, respectively. The differential glycoproteins were mainly involved in various types of N-glycan biosynthesis, mRNA surveillance pathway, and protein processing in the endoplasmic reticulum. These results indicated that an efficient protein homeostasis pathway plays an essential role in the thermophily of C. thermophilum, and N-glycosylation is involved by affecting related proteins. This is the novel study to reveal thermophilic fungi’s physiological response to high-temperature adaptation using omics analysis, facilitating the exploration of the thermophily mechanism of thermophilic fungi.

Improving the thermostability of a thermostable endoglucanase from Chaetomium thermophilum by engineering the conserved noncatalytic residue and N-glycosylation site

Endoglucanases provide an attractive avenue for the bioconversion of lignocellulosic materials into fermentable sugars to supply cellulosic feedstock for biofuels and other value-added chemicals. Thermostable endoglucanases with high catalytic activity are preferred in practical processes. To improve the thermostability and activity of the thermostable β-1,4-endoglucanase CTendo45 isolated from the thermophilic fungus Chaetomium thermophilum, structure-based rational design was performed by using site-directed mutagenesis. When inactivated mutation of the unique N-glycosylation sequon (N88-E89-T90) was implemented and the conserved Y173 residue was substituted with phenylalanine, a double mutant T90A/Y173F demonstrated enzymatic activity that dramatically increased 2.12- and 1.82-fold towards CMC-Na and β-D-glucan, respectively. Additionally, T90A/Y173F exhibited extraordinary heat endurance after 300 min of incubation at elevated temperatures. This study provides a valid approach to the improvement of enzyme redesign protocols and the properties of this endoglucanase mutant distinguish it as an excellent candidate enzyme for industrial biomass conversion.

Insights into the cellulose degradation mechanism of the thermophilic fungus Chaetomium thermophilum based on integrated functional omics

BackgroundLignocellulose is the most abundant and renewable biomass resource on the planet. Lignocellulose can be converted into biofuels and high-value compounds; however, its recalcitrance makes its breakdown a challenge. Lytic polysaccharide monooxygenases (LPMOs) offer tremendous promise for the degradation of recalcitrant polysaccharides. Chaetomium thermophilum, having many LPMO-coding genes, is a dominant thermophilic fungus in cellulose-rich and self-heating habitats. This study explores the genome, secretomes and transcript levels of specific genes of C. thermophilum.ResultsThe genome of C. thermophilum encoded a comprehensive set of cellulose- and xylan-degrading enzymes, especially 18 AA9 LPMOs that belonged to different subfamilies. Extracellular secretomes showed that arabinose and microcrystalline cellulose (MCC) could specifically induce the secretion of carbohydrate-active enzymes (CAZymes), especially AA9 LPMOs, by C. thermophilum under different carbon sources. Temporal analyses of secretomes and transcripts revealed that arabinose induced the secretion of xylanases by C. thermophilum, which was obviously different from other common filamentous fungi. MCC could efficiently induce the specific secretion of LPMO2s, possibly because the insert in loop3 on the substrate-binding surface of LPMO2s strengthened its binding capacity to cellulose. LPMO2s, cellobio hydrolases (CBHs) and cellobiose dehydrogenases (CDHs) were cosecreted, forming an efficient cellulose degradation system of oxidases and hydrolases under thermophilic conditions.ConclusionsThe specific expression of LPMO2s and cosecretion of hydrolases and oxidases by the thermophilic fungus C. thermophilum play an important role in cellulose degradation. This insight increases our understanding of the cellulose degradation under thermophilic conditions and may inspire the design of the optimal enzyme cocktails for more efficient exploration of biomass resources in industrial applications.

Improved Production of Majority Cellulases in Trichoderma reesei by Integration of cbh1 Gene From Chaetomium thermophilum.

Lignocellulose is an abundant waste resource and has been considered as a promising material for production of biofuels or other valuable bio-products. Currently, one of the major bottlenecks in the economic utilization of lignocellulosic materials is the cost-efficiency of converting lignocellulose into soluble sugars for fermentation. One way to address this problem is to seek superior lignocellulose degradation enzymes or further improve current production yields of lignocellulases. In the present study, the lignocellulose degradation capacity of a thermophilic fungus Chaetomium thermophilum was firstly evaluated and compared to that of the biotechnological workhorse Trichoderma reesei. The data demonstrated that compared to T. reesei, C. thermophilum displayed substantially higher cellulose-utilizing efficiency with relatively lower production of cellulases, indicating that better cellulases might exist in C. thermophilum. Comparison of the protein secretome between C. thermophilum and T. reesei showed that the secreted protein categories were quite different in these two species. In addition, to prove that cellulases in C. thermophilum had better enzymatic properties, the major cellulase cellobiohydrolase I (CBH1) from C. thermophilum and T. reesei were firstly characterized, respectively. The data showed that the specific activity of C. thermophilum CBH1 was about 4.5-fold higher than T. reesei CBH1 in a wide range of temperatures and pH. To explore whether increasing CBH1 activity in T. reesei could contribute to improving the overall cellulose-utilizing efficiency of T. reesei, T. reesei cbh1 gene was replaced with C. thermophilum cbh1 gene by integration of C. thermophilum cbh1 gene into T. reesei cbh1 gene locus. The data surprisingly showed that this gene replacement not only increased the cellobiohydrolase activities by around 4.1-fold, but also resulted in stronger induction of other cellulases genes, which caused the filter paper activities, Azo-CMC activities and β-glucosidase activities increased by about 2.2, 1.9, and 2.3-fold, respectively. The study here not only provided new resources of superior cellulases genes and new strategy to improve the cellulase production in T. reesei, but also contribute to opening the path for fundamental research on C. thermophilum.

Molecular Characterization of the HIRA Histone Chaperone Complex from Chaetomium thermophilum

The HIRA histone chaperone complex is an essential epigenetic regulator which deposits the histone variant H3.3 onto DNA to condense it into chromatin and to regulate the epigenetic landscape of the cell. Because the HIRA complex and H3.3 deposition is associated with areas of active transcription in the genome, HIRA dysregulation is associated with disordered development, aging, and maintenance of terminally differentiated cell types. The goal of our studies is to understand the molecular basis of H3.3‐specific histone deposition by the HIRA complex. Toward this goal, we recombinantly express orthologs of the four HIRA complex components‐ HIRA, UBN1, CABIN1, and ASF1a‐ from the thermophilic fungus Chaetomium thermophilum for biochemical and structural characterization. We show efficient recombinant expression of each subunit, determine complex stoichiometry using analytical ultracentrifugation, and begin structural analysis using a combination of X‐ray crystallography and cryo‐electron microscopy of the subunits alone, in complex, and with histones.Support or Funding InformationStructural Biology and Molecular Biophysics Training Program, NIH 5‐T32‐GM‐008275‐29; Epigenetics of Aging and Age‐Associated Diseases P01, NIH 5P01AG031862‐12

Crystal Structure of a GH3 β-Glucosidase from the Thermophilic Fungus Chaetomium thermophilum.

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 Å. 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.

Structural and functional characterization of a frataxin from a thermophilic organism.

Frataxins form an interesting family of iron‐binding proteins with an almost unique fold and are highly conserved from bacteria to primates. They have a pivotal role in iron–sulfur cluster biogenesis as regulators of the rates of cluster formation, as it is testified by the fact that frataxin absence is incompatible with life and reduced levels of the protein lead to the recessive neurodegenerative disease Friedreich's ataxia. Despite its importance, the structure of frataxin has been solved only from relatively few species. Here, we discuss the X‐ray structure of frataxin from the thermophilic fungus Chaetomium thermophilum, and the characterization of its interactions and dynamics in solution. We show that this eukaryotic frataxin has an unusual variation in the classical frataxin fold: the last helix is shorter than in other frataxins which results in a less symmetrical and compact structure. The stability of this protein is comparable to that of human frataxin, currently the most stable among the frataxin orthologues. We also characterized the iron‐binding mode of Ct frataxin and demonstrated that it binds it through a semiconserved negatively charged ridge on the first helix and beta‐strand. Moreover, this frataxin is also able to bind the bacterial ortholog of the desulfurase, which is central in iron–sulfur cluster synthesis, and act as its inhibitor.

Kinetics and Predicted Structure of a Novel Xylose Reductase from Chaetomium thermophilum.

While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable.

Structural and biochemical analysis of a NOT1 MIF4G-like domain of the CCR4-NOT complex

The CCR4-NOT complex plays a central role in the regulation of gene expression and degradation of messenger RNAs. The multisubunit complex assembles on the NOT1 protein, which acts as a ‘scaffold’ and is highly conserved in eukaryotes. NOT1 consists of a series of helical domains that serve as docking sites for other CCR4-NOT subunits. We describe a crystal structure of a connector domain of NOT1 from the thermophilic fungus Chaetomium thermophilum (Ct). Comparative structural analysis indicates that this domain adopts a MIF4G-like fold and we have termed it the MIF4G-C domain. Solution scattering studies indicate that the human MIF4G-C domain likely adopts a very similar fold to the Ct MIF4G-C. MIF4G domains have been described to mediate interactions with DEAD-box helicases such as DDX6. However, comparison of the interfaces of the MIF4G-C with the MIF4G domain of NOT1 that interacts with DDX6 reveals key structural differences that explain why the MIF4G-C does not bind DDX6. We further show that the human MIF4G-C does not interact stably with other subunits of the CCR4-NOT complex. The structural conservation of the MIF4G-C domain suggests that it may have an important but presently undefined role in the CCR4-NOT complex.

Reconstitution of a thermophilic Cu+ importer in vitro reveals intrinsic high-affinity slow transport driving accumulation of an essential metal ion

Acquisition of the trace element copper (Cu) is critical to drive essential eukaryotic processes such as oxidative phosphorylation, iron mobilization, peptide hormone biogenesis, and connective tissue maturation. The Ctr1/Ctr3 family of Cu importers, first discovered in fungi and conserved in mammals, are critical for Cu+ movement across the plasma membrane or mobilization from endosomal compartments. Whereas ablation of Ctr1 in mammals is embryonic lethal, and Ctr1 is critical for dietary Cu absorption, cardiac function, and systemic iron distribution, little is known about the intrinsic contribution of Ctr1 for Cu+ permeation through membranes or its mechanism of action. Here, we identify three members of a Cu+ importer family from the thermophilic fungus Chaetomium thermophilum: Ctr3a and Ctr3b, which function on the plasma membrane, and Ctr2, which likely functions in endosomal Cu mobilization. All three proteins drive Cu and isoelectronic silver (Ag) uptake in cells devoid of Cu+ importers. Transport activity depends on signature amino acid motifs that are conserved and essential for all Ctr1/3 transporters. Ctr3a is stable and amenable to purification and was incorporated into liposomes to reconstitute an in vitro Ag+ transport assay characterized by stopped-flow spectroscopy. Ctr3a has intrinsic high-affinity metal ion transport activity that closely reflects values determined in vivo, with slow turnover kinetics. Given structural models for mammalian Ctr1, Ctr3a likely functions as a low-efficiency Cu+ ion channel. The Ctr1/Ctr3 family may be tuned to import essential yet potentially toxic Cu+ ions at a slow rate to meet cellular needs, while minimizing labile intracellular Cu+ pools.

Expression, Functional Characterization, and Preliminary Crystallization of the Cochaperone Prefoldin from the Thermophilic Fungus Chaetomium thermophilum.

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits, and has the appearance of a jellyfish: Its body consists of a double β-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. Based on structural information from archaeal prefoldins, mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the structure and mechanisms of eukaryotic prefoldins remain unknown. In this study, we succeeded in obtaining recombinant prefoldin from a thermophilic fungus, Chaetomium thermophilum (CtPFD). The recombinant CtPFD could not protect citrate synthase from thermal aggregation. However, CtPFD formed a complex with actin from chicken muscle and tubulin from porcine brain, suggesting substrate specificity. We succeeded in observing the complex formation of CtPFD and the group II chaperonin of C. thermophilum (CtCCT) by atomic force microscopy and electron microscopy. These interaction kinetics were analyzed by surface plasmon resonance using Biacore. Finally, we have shown the transfer of actin from CtPFD to CtCCT. The study of the folding pathway formed by CtPFD and CtCCT should provide important information on mechanisms of the eukaryotic prefoldin–chaperonin system.

Discovery and engineering of enhanced SUMO protease enzymes

Small ubiquitin-like modifier (SUMO) is commonly used as a protein fusion domain to facilitate expression and purification of recombinant proteins, and a SUMO-specific protease is then used to remove SUMO from these proteins. Although this protease is highly specific, its limited solubility and stability hamper its utility as an in vitro reagent. Here, we report improved SUMO protease enzymes obtained via two approaches. First, we developed a computational method and used it to re-engineer WT Ulp1 from Saccharomyces cerevisiae to improve protein solubility. Second, we discovered an improved SUMO protease via genomic mining of the thermophilic fungus Chaetomium thermophilum, as proteins from thermophilic organisms are commonly employed as reagent enzymes. Following expression in Escherichia coli, we found that these re-engineered enzymes can be more thermostable and up to 12 times more soluble, all while retaining WT-or-better levels of SUMO protease activity. The computational method we developed to design solubility-enhancing substitutions is based on the RosettaScripts application for the macromolecular modeling suite Rosetta, and it is broadly applicable for the improvement of solution properties of other proteins. Moreover, we determined the X-ray crystal structure of a SUMO protease from C. thermophilum to 1.44 Å resolution. This structure revealed that this enzyme exhibits structural and functional conservation with the S. cerevisiae SUMO protease, despite exhibiting only 28% sequence identity. In summary, by re-engineering the Ulp1 protease and discovering a SUMO protease from C. thermophilum, we have obtained proteases that are more soluble, more thermostable, and more efficient than the current commercially available Ulp1 enzyme.

An Evolutionarily Conserved Structural Platform for PRC2 Inhibition by a Class of Ezh2 Inhibitors

Polycomb repressive complex 2 (PRC2) mediates trimethylation of histone H3K27 (H3K27me3), an epigenetic hallmark for repressed chromatin. Overactive mutants of the histone lysine methyltransferase subunit of PRC2, Ezh2, are found in various types of cancers. Pyridone-containing inhibitors such as GSK126 compete with S-adenosylmethionine (SAM) for Ezh2 binding and effectively inhibit PRC2 activity. PRC2 from the thermophilic fungus Chaetomium thermophilum (ct) is functionally similar to the human version in several regards and has the added advantage of producing high-resolution crystal structures, although inhibitor-bound structures of human or human/chameleon PRC2 are also available at up to 2.6 Å resolution. We solved crystal structures of both human and ctPRC2 bound to GSK126 and the structurally similar inhibitor GSK343. While the two organisms feature a disparate degree of inhibitor potency, surprisingly, GSK126 binds in a similar manner in both structures. Structure-guided protein engineering of the drug binding pocket allowed us to introduce humanizing mutations into ctEzh2 to produce a ctPRC2 variant that is more susceptible to GSK126 inhibition. Additional analysis indicated that an evolutionarily conserved structural platform dictates a unique mode of GSK126 binding, suggesting a mechanism of drug selectivity. The existing drug scaffold may thus be used to probe the function and cellular regulation of PRC2 in a wide spectrum of organisms, ranging from fungi to humans.

Characterization of a novel thermostable GH7 endoglucanase from Chaetomium thermophilum capable of xylan hydrolysis

A new endoglucanase encoding gene (ctendo7) was cloned from the thermophilic fungus Chaetomium thermophilum and heterologously expressed in Pichia pastoris. The recombinant CTendo7 enzyme was purified by Ni2+ affinity chromatography and subsequently characterized. CTendo7 belongs to glycoside hydrolase family 7, and exhibited considerable activity against sodium carboxymethyl cellulose (CMC-Na) and xylan of 1.91 IU/mg and 3.05 IU/mg at the optimum reaction condition of 55 °C, pH 5.0, respectively. The purified enzyme displayed relatively good thermostability. The residual endoglucanase and xylanase activities were 74.3% and 66.2% after a 60 min pre-incubation at 70 °C. Additionally, Ag+, Fe3+ and Cu2+ negatively affected the enzyme's activity, while the presence of 1 mM and 5 mM Mn2+ significantly enhanced both endoglucanase and xylanase activities. Generation of soluble oligosaccharides from lignocellulose is a critical step in bioethanol production, and it is noteworthy that CTendo7 produced cello-oligosaccharides and xylo-oligosaccharides from the continuous enzymatic saccharification of CMC-Na and xylan, respectively. This is the first detailed report on a novel bifunctional endoglucanase/xylanase enzyme from C. thermophilum. Furthermore, the excellent properties of CTendo7 distinguish it as a promising candidate for industrial lignocellulosic biomass conversion.