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.

PRODUCTION OF ENZYMES IN PREDOMINANT THERMOPHILIC FUNGI AVAILABLE FROM ORGANIC SUBSTRATES

Present investigation describes that the study site comes under Aurangabad Division Maharashtra and it falls in Deccan Plateau Zone of India. It was collected different types of organic substrates viz. vermiompost, poultary manure, baggase, farm yard manure (FYM), soil, Ash etc. Isolated thermophilic predominant fungi thermophilic fungi viz.Aspergillus niger, Mucor mucedo,Humicola insolens,Trichoderma harzianum,T. viride,Penicillium duponti,Fusarium oxysporun and Chaetomium thermophilum were carried out for the production of enzymes. Isolated predominant thermophilic fungi were evaluated on different types of enzymes. Among tested thermophilic fungi, the highest ativity was observed in C. thermophilium (20mm) followed by T. harzianum (19.50mm) In lipase, M. mucedo (15.40mm) was found maximum followed by F. oxysporun. Cellulase activity was found highest in A. nige (25mm) followed by others. In case of xylanase, catalase, peroxidase and esterase activities were found maximum, minimum and medium even negative in some fungi. Maximum pectinase activity was detected from H. insolens (52.26 @ 0 min) and (74.25 @ 10 min) and in case of M. mucedo, F. oxysporun and C. thermophilium was found most extreme while least in A. niger (30.12) and P. duponti (33.47) @ 0 minute. Key words: Organic Substrates, Thermophilic Fungi, Enzymes

Global Transcriptome Characterization and Assembly of the Thermophilic Ascomycete Chaetomium thermophilum

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.

Cesium based phasing of macromolecules: a general easy to use approach for solving the phase problem

Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. However, solving the phase problem is still one of the biggest obstacles on the way of successfully determining a crystal structure. To overcome this caveat, we have utilized the anomalous scattering properties of the heavy alkali metal cesium. We investigated the introduction of cesium in form of cesium chloride during the three major steps of protein treatment in crystallography: purification, crystallization, and cryo-protection. We derived a step-wise procedure encompassing a “quick-soak”-only approach and a combined approach of CsCl supplement during purification and cryo-protection. This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from Chaetomium thermophilum for de novo structure determination. Usage of CsCl thus provides a versatile, general, easy to use, and low cost phasing strategy.

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.

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

The synchrotron facilities used in collecting the data for the article by Švecová et al. [(2021), Acta Cryst. D77, 755–775] are acknowledged.

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.

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.