Gcn2 structurally mimics and functionally repurposes the HisRS enzyme for the integrated stress response.

Protein kinase Gcn2 attenuates protein synthesis in response to amino acid starvation while stimulating translation of a transcriptional activator of amino acid biosynthesis. Gcn2 activation requires a domain related to histidyl-tRNA synthetase (HisRS), the enzyme that aminoacylates tRNAHis. While evidence suggests that deacylated tRNA binds the HisRS domain for kinase activation, ribosomal P-stalk proteins have been implicated as alternative activating ligands on stalled ribosomes. We report crystal structures of the HisRS domain of Chaetomium thermophilum Gcn2 that reveal structural mimicry of both catalytic (CD) and anticodon-binding (ABD) domains, which in authentic HisRS bind the acceptor stem and anticodon loop of tRNAHis. Elements for forming histidyl adenylate and aminoacylation are lacking, suggesting that Gcn2HisRS was repurposed for kinase activation, consistent with mutations in the CD that dysregulate yeast Gcn2 function. Substituting conserved ABD residues well positioned to contact the anticodon loop or that form a conserved ABD-CD interface impairs Gcn2 function in starved cells. Mimicry in Gcn2HisRS of two highly conserved structural domains for binding both ends of tRNA-each crucial for Gcn2 function-supports that deacylated tRNAs activate Gcn2 and exemplifies how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function.

Structural and functional insights underlying recognition of histidine phosphotransfer protein in fungal phosphorelay systems.

In human pathogenic fungi, receiver domains from hybrid histidine kinases (hHK) have to recognize one HPt. To understand the recognition mechanism, we have assessed phosphorelay from receiver domains of five hHKs of group III, IV, V, VI, and XI to HPt from Chaetomium thermophilum and obtained the structures of Ct_HPt alone and in complex with the receiver domain of hHK group VI. Our data indicate that receiver domains phosphotransfer to Ct_HPt, show a low affinity for complex formation, and prevent a Leu-Thr switch to stabilize phosphoryl groups, also derived from the structures of the receiver domains of hHK group III and Candida albicans Sln1. Moreover, we have elucidated the envelope structure of C. albicans Ypd1 using small-angle X-ray scattering which reveals an extended flexible conformation of the long loop αD-αE which is not involved in phosphotransfer. Finally, we have analyzed the role of salt bridges in the structure of Ct_HPt alone.

Kinetic and structural insights into the requirement of fungal tRNA ligase for a 2'-phosphate end.

Fungal RNA ligase (LIG) is an essential tRNA splicing enzyme that joins 3'-OH,2'-PO4 and 5'-PO4 RNA ends to form a 2'-PO4,3'-5' phosphodiester splice junction. Sealing entails three divalent cation-dependent adenylate transfer steps. First, LIG reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and displace pyrophosphate. Second, LIG transfers AMP to the 5'-PO4 RNA terminus to form an RNA-adenylate intermediate (A5'pp5'RNA). Third, LIG directs the attack of an RNA 3'-OH on AppRNA to form the splice junction and displace AMP. A defining feature of fungal LIG vis-à-vis canonical polynucleotide ligases is the requirement for a 2'-PO4 to synthesize a 3'-5' phosphodiester bond. Fungal LIG consists of an N-terminal adenylyltransferase domain and a unique C-terminal domain. The C-domain of Chaetomium thermophilum LIG (CthLIG) engages a sulfate anion thought to be a mimetic of the terminal 2'-PO4 Here, we interrogated the contributions of the C-domain and the conserved sulfate ligands (His227, Arg334, Arg337) to ligation of a pRNA2'p substrate. We find that the C-domain is essential for end-joining but dispensable for ligase adenylylation. Mutations H227A, R334A, and R337A slowed the rate of step 2 RNA adenylation by 420-fold, 120-fold, and 60-fold, respectively, vis-à-vis wild-type CthLIG. An R334A-R337A double-mutation slowed step 2 by 580-fold. These results fortify the case for the strictly conserved His-Arg-Arg triad as the enforcer of the 2'-PO4 end-specificity of fungal tRNA ligases and as a target for small molecule interdiction of fungal tRNA splicing.

Structural basis of S-adenosylmethionine-dependent allosteric transition from active to inactive states in methylenetetrahydrofolate reductase.

Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation and S-adenosylmethionine (AdoMet)-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures of Chaetomium thermophilum MTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form of cMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites in hMTHFR, three of which were previously unidentified. Based on the structural framework provided by our cMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition.

Activation of secondary metabolite gene clusters in Chaetomium olivaceum via the deletion of a histone deacetylase.

Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary metabolite (SM) biosynthetic gene clusters (BGCs). Histone deacetylases (HDACs) play a pivotal role in determining the extent of histone acetylation modifications and act as triggers for the expression activity of target BGCs. The genus Chaetomium is widely recognized as a rich source of novel and bioactive SMs. Deletion of a class I HDAC gene of Chaetomium olivaceum SD-80A, g7489, induces a substantial pleiotropic effect on the expression of SM BGCs. The C. olivaceum SD-80A ∆g7489 strain exhibited significant changes in morphology, sporulation ability, and secondary metabolic profile, resulting in the emergence of new compound peaks. Notably, three polyketides (A1-A3) and one asterriquinone (A4) were isolated from this mutant strain. Furthermore, our study explored the BGCs of A1-A4, confirming the function of two polyketide synthases (PKSs). Collectively, our findings highlight the promising potential of molecular epigenetic approaches for the elucidation of novel active compounds and their biosynthetic elements in Chaetomium species. This finding holds great significance for the exploration and utilization of Chaetomium resources. KEY POINTS: • Deletion of a class I histone deacetylase activated secondary metabolite gene clusters. • Three polyketides and one asterriquinone were isolated from HDAC deleted strain. • Two different PKSs were reported in C. olivaceum SD-80A.

Atg15 is a vacuolar phospholipase that disintegrates organelle membranes.

Atg15 (autophagy-related 15) is a vacuolar phospholipase essential for the degradation of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies, hereinafter referred to as intravacuolar/intralysosomal autophagic compartments (IACs), but it remains unknown if Atg15 directly disrupts IAC membranes. Here, we show that the recombinant Chaetomium thermophilum Atg15 lipase domain (CtAtg15(73-475)) possesses phospholipase activity. The activity of CtAtg15(73-475) was markedly elevated by limited digestion. We inserted the human rhinovirus 3C protease recognition sequence and found that cleavage between S159 and V160 was important to activate CtAtg15(73-475). Our molecular dynamics simulation suggested that the cleavage facilitated conformational change around the active center of CtAtg15, resulting in an exposed state. We confirmed that CtAtg15 could disintegrate S. cerevisiae IAC in vivo. Further, both mitochondria and IAC of S. cerevisiae were disintegrated by CtAtg15. This study suggests Atg15 plays a role in disrupting any organelle membranes delivered to vacuoles by autophagy.

Purification and anticancer activity of glutaminase and urease free intracellular l-asparaginase from Chaetomium sp.

l-asparaginase is a chemotherapeutic drug used in the treatment of acute lymphoblastic leukemia, a malignant disorder in children. l-asparaginase helps in removing acrylamide found in fried and baked foods which is carcinogenic in nature. The search for new therapeutic enzymes is of great interest in both medical and food applications. The present work aims to isolate the intracellular l-asparaginase from endophytic fungi Chaetomium sp. The intracellular enzyme was partially purified by chromatographic techniques. Molecular weight of enzyme was found to be ~66 kDa by SDS PAGE analysis. The enzyme is highly specific for l-asparagine and did not show glutaminase and urease activity. Maximum enzyme activity was found to be 58 ± 5 U/mL at 40 °C, pH 7.0 with 2 µg of protein. Intracellular l-asparaginase from Chaetomium sp. exhibited anticancer activity on human blood cancer (MOLT-4) cells.

Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction.

Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture.

Effect of carbohydrate binding modules alterations on catalytic activity of glycoside hydrolase family 6 exoglucanase from Chaetomium thermophilum to cellulose.

Exoglucanase (CBH) is the rate limiting enzyme in the process of cellulose degradation. The carbohydrate binding module (CBM) can improve the accessibility of cellulase to substrate, thereby promoting the enzymatic hydrolysis of cellulase. In this study, the influence of CBM on the properties of GH6 exoglucanase from Chaetomium thermophilum (CtCBH) is systematically explored from three perspectives: the fusion of exogenous CBM, the exogenous CBM replacement of its own CBM, and the deletion of its own CBM. The parental and reconstructed CtCBH presented the same optimum pH (6.0) and temperature (60 °C) for maximum activity. Fusion of exogenous CBM increased the binding capacity of CtCBH to Avicel by 8% and 9%, respectively, but it had no significant effect on its catalytic activity. The exogenous CBM replacement of its own CBM resulted in a 12% reduction in the binding ability of CtCBH to Avicel, and a 26% reduction in the catalytic activity of Avicel. The deletion of its own CBM significantly reduced the binding ability of CtCBH to Avicel by approximately 53%, but its catalytic activity was not obviously reduced. These observations suggest that binding ability of CBM is not necessary for the catalysis of CtCBH.

Crystallographic fragment screening-based study of a novel FAD-dependent oxidoreductase from Chaetomium thermophilum.

The FAD-dependent oxidoreductase from Chaetomium thermophilum (CtFDO) is a novel thermostable glycoprotein from the glucose-methanol-choline (GMC) oxidoreductase superfamily. However, CtFDO shows no activity toward the typical substrates of the family and high-throughput screening with around 1000 compounds did not yield any strongly reacting substrate. Therefore, protein crystallography, including crystallographic fragment screening, with 42 fragments and 37 other compounds was used to describe the ligand-binding sites of CtFDO and to characterize the nature of its substrate. The structure of CtFDO reveals an unusually wide-open solvent-accessible active-site pocket with a unique His-Ser amino-acid pair putatively involved in enzyme catalysis. A series of six crystal structures of CtFDO complexes revealed five different subsites for the binding of aryl moieties inside the active-site pocket and conformational flexibility of the interacting amino acids when adapting to a particular ligand. The protein is capable of binding complex polyaromatic substrates of molecular weight greater than 500 Da.

Structural characterisation of the Chaetomium thermophilum Chl1 helicase.

Chl1 is a member of the XPD family of 5'-3' DNA helicases, which perform a variety of roles in genome maintenance and transmission. They possess a variety of unique structural features, including the presence of a highly variable, partially-ordered insertion in the helicase domain 1. Chl1 has been shown to be required for chromosome segregation in yeast due to its role in the formation of persistent chromosome cohesion during S-phase. Here we present structural and biochemical data to show that Chl1 has the same overall domain organisation as other members of the XPD family, but with some conformational alterations. We also present data suggesting the insert domain in Chl1 regulates its DNA binding.

Enhancing recombinant Chaetomium thermophilium Formate Dehydrogenase Expression with CRISPR Technology.

Genetic manipulation of Escherichia coli influences the regulation of bacterial metabolism, which could be useful for the production of different targeted products. The RpoZ gene encodes for the ω subunit of the RNA polymerase (RNAP) and is involved in the regulation of the relA gene pathway. RelA is responsible for the production of guanosine pentaphosphate (ppGpp), which is a major alarmone in the stringent response. Expression of relA is reduced in the early hours of growth of RpoZ mutant E. coli. In the absence of the ω subunit, ppGpp affinity to RNAP is decreased; thus, rpoZ gene deleted E. coli strains show a modified stringent response. We used the E. coli K-12 MG1655 strain that lacks rpoZ (JEN202) to investigate the effect of the modified stringent response on recombinant protein production. However, the absence of the ω subunit results in diminished stability of the RNA polymerase at the promoter site. To avoid this, we used a deactivated CRISPR system that targets the ω subunit to upstream of the promoter site in the expression plasmid. The expression plasmid encodes for Chaetomium thermophilum formate dehydrogenase (CtFDH), a valuable enzyme for cofactor regeneration and CO2 reduction. A higher amount of CtFDH from the soluble fraction was purified from the JEN202 strain compared to the traditional BL21(DE3) method, thus offering a new strategy for batch-based recombinant enzyme production.

The structure of Prp2 bound to RNA and ADP-BeF3- reveals structural features important for RNA unwinding by DEAH-box ATPases.

Noncoding intron sequences present in precursor mRNAs need to be removed prior to translation, and they are excised via the spliceosome, a multimegadalton molecular machine composed of numerous protein and RNA components. The DEAH-box ATPase Prp2 plays a crucial role during pre-mRNA splicing as it ensures the catalytic activation of the spliceosome. Despite high structural similarity to other spliceosomal DEAH-box helicases, Prp2 does not seem to function as an RNA helicase, but rather as an RNA-dependent ribonucleoprotein particle-modifying ATPase. Recent crystal structures of the spliceosomal DEAH-box ATPases Prp43 and Prp22, as well as of the related RNA helicase MLE, in complex with RNA have contributed to a better understanding of how RNA binding and processivity might be achieved in this helicase family. In order to shed light onto the divergent manner of function of Prp2, an N-terminally truncated construct of Chaetomium thermophilum Prp2 was crystallized in the presence of ADP-BeF3- and a poly-U12 RNA. The refined structure revealed a virtually identical conformation of the helicase core compared with the ADP-BeF3-- and RNA-bound structure of Prp43, and only a minor shift of the C-terminal domains. However, Prp2 and Prp43 differ in the hook-loop and a loop of the helix-bundle domain, which interacts with the hook-loop and evokes a different RNA conformation immediately after the 3' stack. On replacing these loop residues in Prp43 by the Prp2 sequence, the unwinding activity of Prp43 was abolished. Furthermore, a putative exit tunnel for the γ-phosphate after ATP hydrolysis could be identified in one of the Prp2 structures.

Crystal Structure of a Cu,Zn Superoxide Dismutase From the Thermophilic Fungus Chaetomium thermophilum.

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. METHODS: 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 Å 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.

Integrative structure of a 10-megadalton eukaryotic pyruvate dehydrogenase complex from native cell extracts.

The pyruvate dehydrogenase complex (PDHc) is a giant enzymatic assembly involved in pyruvate oxidation. PDHc components have been characterized in isolation, but the complex's quaternary structure has remained elusive due to sheer size, heterogeneity, and plasticity. Here, we identify fully assembled Chaetomium thermophilum α-keto acid dehydrogenase complexes in native cell extracts and characterize their domain arrangements utilizing mass spectrometry, activity assays, crosslinking, electron microscopy (EM), and computational modeling. We report the cryo-EM structure of the PDHc core and observe unique features of the previously unknown native state. The asymmetric reconstruction of the 10-MDa PDHc resolves spatial proximity of its components, agrees with stoichiometric data (60 E2p:12 E3BP:∼20 E1p: ≤ 12 E3), and proposes a minimum reaction path among component enzymes. The PDHc shows the presence of a dynamic pyruvate oxidation compartment, organized by core and peripheral protein species. Our data provide a framework for further understanding PDHc and α-keto acid dehydrogenase complex structure and function.

Structural basis of Naa20 activity towards a canonical NatB substrate.

N-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development.

Clamping, bending, and twisting inter-domain motions in the misfold-recognizing portion of UDP-glucose: Glycoprotein glucosyltransferase.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (CtUGGT), two CtUGGT double-cysteine mutants, and its TRXL2 domain truncation (CtUGGT-ΔTRXL2). CtUGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name "Parodi limit" the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro, in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70-80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a "one-size-fits-all adjustable spanner" UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain.

Food-Grade Expression and Characterization of a Dextranase from Chaetomium gracile Suitable for Sugarcane Juice Clarification.

The microbial production of dextranase using cheap carbon sources is beneficial to solve the economic loss caused by the accumulation of dextran in syrup. A food-grade microbial cell factory was constructed by introducing the dextranase encoding gene DEX from Chaetomium gracile to the chromosome of Bacillus subtilis, and the antibiotic resistance marker gene was subsequently deleted via the Cre/loxP strategy. The dual-promoter system with a sequentially arranged constitutive P43 promoter resulted in an 85 % increase in DEX expression. Under the optimal fermentation conditions of 10 g/L maltose, 15 g/L casein, 1 g/L Na2 HPO4 , 1 g/L FeSO4 and 8 g/L NaCl, DEX activity was increased from 2.625 to 64.34 U/mL. Recombinant DEX was purified 5.98-fold with a recovery ratio of 26.67 % and specific activity of 3935.02 U/mg. Enzyme activity was optimal at 55 °C and pH 5.0 and remained 80.34 % and 71.36 % of the initial activity at 55 °C and pH 4.0 after 60 min, respectively. The enzyme possessed high activity in the presence of Co2+ , while Ag+ showed the strongest inhibition ability. The optimal substrate was 20 g/L dextran T-2000. The findings could facilitate the low-cost, large-scale production of food-grade DEX for use in the sugar industry.

High-level expression and enzymatic properties of a novel thermostable xylanase with high arabinoxylan degradation ability from Chaetomium sp. suitable for beer mashing.

A novel thermostable xylanase gene from Chaetomium sp. CQ31 was cloned and codon-optimized (CsXynBop). The deduced protein sequence of the gene shared the highest similarity of 75% with the glycoside hydrolase (GH) family 10 xylanase from Achaetomium sp. Xz-8. CsXynBop was over-expressed in Pichia pastoris GS115 by high-cell density fermentation, with the highest xylanase yield of 10,017 U/mL. The recombinant xylanase (CsXynBop) was purified to homogeneity and biochemically characterized. CsXynBop was optimally active at pH 6.5 and 85 °C, respectively, and stable over a broad pH range of 5.0-9.5 and up to 60 °C. The enzyme exhibited strict substrate specificity towards oat-spelt xylan (2, 489 U/mg), beechwood xylan (1522 U/mg), birchwood xylan (1067 U/mg), and showed relatively high activity towards arabinoxylan (1208 U/mg), but exhibited no activity on other tested polysaccharides. CsXynBop hydrolyzed different xylans to yield mainly xylooligosaccharides (XOSs) with degree of polymerization (DP) 2-5. The application of CsXynBop (200 U/g malt) in malt mashing substantially decreased the filtration time and viscosity of malt by 42.3% and 8.6%, respectively. These excellent characteristics of CsXynBop may make it a good candidate in beer industry.

Structural Characterization of Glycerol Kinase from the Thermophilic Fungus Chaetomium thermophilum.

Glycerol is an organic compound that can be utilized as an alternative source of carbon by various organisms. One of the ways to assimilate glycerol by the cell is the phosphorylative catabolic pathway in which its activation is catalyzed by glycerol kinase (GK) and glycerol-3-phosphate (G3P) is formed. To date, several GK crystal structures from bacteria, archaea, and unicellular eukaryotic parasites have been solved. Herein, we present a series of crystal structures of GK from Chaetomium thermophilum (CtGK) in apo and glycerol-bound forms. In addition, we show the feasibility of an ADP-dependent glucokinase (ADPGK)-coupled enzymatic assay to measure the CtGK activity. New structures described in our work provide structural insights into the GK catalyzed reaction in the filamentous fungus and set the foundation for understanding the glycerol metabolism in eukaryotes.