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Glycoside hydrolases (GHs) are enzymes involved in the degradation of oligosaccharides and polysaccharides. The sequence space of GHs is rapidly expanding due to the increasing number of available sequences. This expansion paves the way for the discovery of novel enzymes with peculiar structural and functional properties. This work is focused on two GHs, Ps_GH5 and Ps_GH50, from the genome of the Antarctic bacterium Pseudomonas sp. ef1. These enzymes are in an unexplored region of the sequence space of their respective GH families, not allowing a reliable sequence-based function prediction. For this reason, a computational pipeline was developed that combines deep learning "dynamic docking" on AlphaFold 3D models with physics-based molecular dynamics simulations to infer their substrate specificity. From in silico screening of a repertoire of potential oligosaccharides, only xylooligosaccharides for Ps_GH5 and galactooligosaccharides for Ps_GH50 emerged as catalytically competent substrates. Biochemical characterization agrees with computational simulations indicating that Ps_GH5 is an endo-β-xylanase, and Ps_GH50 is active mainly on small galactooligosaccharides. In conclusion, this study identifies two novel GHs subfamilies placed in remote regions of the sequence space and highlights the efficacy of substrate specificity prediction by computational approaches in the discovery of new enzymes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Marco Mangiagalli reports financial support was provided by University of Milan-Bicocca. Marina Lotti reports financial support was provided by University of Milan-Bicocca. Salvatore Fusco reports was provided by Ministero dell'Università e della Ricerca (MUR). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Author(s). Published by Elsevier B.V. All rights reserved.)

The rise of agro-industrial activities over recent decades has exponentially increased lignocellulose biomasses (LCB) production. LCB serves as a cost-effective source for fermentable sugars and other renewable chemicals. This study explores the use of microbial consortia, particularly thermophilic consortia, for LCB deconstruction. Thermophiles produce stable enzymes that retain activity under industrial conditions, presenting a promising approach for LCB conversion. This research focused on two microbial consortia (i.e., microbiomes) that were analyzed for enzyme production using a cheap medium, i.e., a mixture of spent mushroom substrate (SMS) and digestate. The secreted xylanolytic enzymes were characterized in terms of temperature and pH optima, thermal stability, and hydrolysis products from LCB-derived polysaccharides. These enzymes showed optimal activity aligning with common biorefinery conditions and outperformed a formulated enzyme mixture in thermostability tests in the digestate. Phylogenetic and genomic analyses highlighted the genetic diversity and metabolic potential of these microbiomes. Bacillus licheniformis was identified as a key species, with two distinct strains contributing to enzyme production. The presence of specific glycoside hydrolases involved in the cellulose and hemicellulose degradation underscores these consortia's capacity for efficient LCB conversion. These findings highlight the potential of thermophilic microbiomes, isolated from an industrial environment, as a robust source of robust enzymes, paving the way for more sustainable and cost-effective bioconversion processes in biofuel and biochemical production and other biotechnological applications.

Glycoside hydrolases (GHs) are pivotal in the hydrolysis of the glycosidic bonds of sugars, which are the main carbon and energy sources. The genome of Marinomonas sp. ef1, an Antarctic bacterium, contains three GHs belonging to family 3. These enzymes have distinct architectures and low sequence identity, suggesting that they originated from separate horizontal gene transfer events. M-GH3&#95;A and M-GH3&#95;B, were found to differ in cold adaptation and substrate specificity. M-GH3&#95;A is a bona fide cold-active enzyme since it retains 20 % activity at 10 °C and exhibits poor long-term thermal stability. On the other hand, M-GH3&#95;B shows mesophilic traits with very low activity at 10 °C (< 5 %) and higher long-term thermal stability. Substrate specificity assays highlight that M-GH3&#95;A is a promiscuous β-glucosidase mainly active on cellobiose and cellotetraose, whereas M-GH3&#95;B is a β-xylosidase active on xylan and arabinoxylan. Structural analysis suggests that such functional differences are due to their differently shaped active sites. The active site of M-GH3&#95;A is wider but has a narrower entrance compared to that of M-GH3&#95;B. Genome-based prediction of metabolic pathways suggests that Marinomonas sp. ef1 can use monosaccharides derived from the GH3-catalyzed hydrolysis of oligosaccharides either as a carbon source or for producing osmolytes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Marco Mangiagalli reports financial support was provided by University of Milano-Bicocca. Marina Lotti reports financial support was provided by University of Milano-Bicocca. Salvatore Fusco reports financial support was provided by University of Verona. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Lignocellulose biomasses (LCB), including spent mushroom substrate (SMS), pose environmental challenges if not properly managed. At the same time, these renewable resources hold immense potential for biofuel and chemicals production. With the mushroom market growth expected to amplify SMS quantities, repurposing or disposal strategies are critical. This study explores the use of SMS for cultivating microbial communities to produce carbohydrate-active enzymes (CAZymes). Addressing a research gap in using anaerobic digesters for enriching microbiomes feeding on SMS, this study investigates microbial diversity and secreted CAZymes under varied temperatures (37 °C, 50 °C, and 70 °C) and substrates (SMS as well as pure carboxymethylcellulose, and xylan). Enriched microbiomes demonstrated temperature-dependent preferences for cellulose, hemicellulose, and lignin degradation, supported by thermal and elemental analyses. Enzyme assays confirmed lignocellulolytic enzyme secretion correlating with substrate degradation trends. Notably, thermogravimetric analysis (TGA), coupled with differential scanning calorimetry (TGA-DSC), emerged as a rapid approach for saccharification potential determination of LCB. Microbiomes isolated at mesophilic temperature secreted thermophilic hemicellulases exhibiting robust stability and superior enzymatic activity compared to commercial enzymes, aligning with biorefinery conditions. PCR-DGGE and metagenomic analyses showcased dynamic shifts in microbiome composition and functional potential based on environmental conditions, impacting CAZyme abundance and diversity. The meta-functional analysis emphasised the role of CAZymes in biomass transformation, indicating microbial strategies for lignocellulose degradation. Temperature and substrate specificity influenced the degradative potential, highlighting the complexity of environmental-microbial interactions. This study demonstrates a temperature-driven microbial selection for lignocellulose degradation, unveiling thermophilic xylanases with industrial promise. Insights gained contribute to optimizing enzyme production and formulating efficient biomass conversion strategies. Understanding microbial consortia responses to temperature and substrate variations elucidates bioconversion dynamics, emphasizing tailored strategies for harnessing their biotechnological potential.

Centrins are calcium (Ca 2+ )-binding proteins that are involved in many cellular functions including centrosome regulation. A known cellular target of centrins is SFI1, a large centrosomal protein containing multiple repeats that represent centrin-binding motifs. Recently, a protein homologous to yeast and mammalian SFI1, denominated TgSFI1, which shares SFI1-repeat organization, was shown to colocalize at centrosomes with centrin 1 from Toxoplasma gondii (TgCEN1). However, the molecular details of the interaction between TgCEN1 and TgSFI1 remain largely unknown. Herein, combining different biophysical methods, including isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy, we determined the binding properties of TgCEN1 and its individual N- and C-terminal domains to synthetic peptides derived from distinct repeats of TgSFI1. Overall, our data indicate that the repeats in TgSFI1 constitute binding sites for TgCEN1, but the binding modes of TgCEN1 to the repeats differ appreciably in terms of binding affinity, Ca 2+ sensitivity, and lobe-specific interaction. These results suggest that TgCEN1 displays remarkable conformational plasticity, allowing for the distinct repeats in TgSFI1 to possess precise modes of TgCEN1 binding and regulation during Ca 2+ sensing, which appears to be crucial for the dynamic association of TgCEN1 with TgSFI1 in the centrosome architecture.

Centrins are a family of small, EF hand-containing proteins that are found in all eukaryotes and are often complexed with centrosome-related structures. Since their discovery, centrins have attracted increasing interest due to their multiple, diverse cellular functions. Centrins are similar to calmodulin (CaM) in size, structure and domain organization, although in contrast to CaM, the majority of centrins possess at least one calcium (Ca 2+ ) binding site that is non-functional, thus displaying large variance in Ca 2+ sensing abilities that could support their functional versatility. In this review, we summarize current knowledge on centrins from both biophysical and structural perspectives with an emphasis on centrin-target interactions. In-depth analysis of the Ca 2+ sensing properties of centrins and structures of centrins complexed with target proteins can provide useful insight into the mechanisms of the different functions of centrins and how these proteins contribute to the complexity of the Ca 2+ signaling cascade. Moreover, it can help to better understand the functional redundancy of centrin isoforms and centrin-binding proteins.

Centrins are conserved calcium (Ca2+)-binding proteins typically associated with centrosomes that have been implicated in several biological processes. In Toxoplasma gondii, a parasite that causes toxoplasmosis, three centrin isoforms have been recognized. We have recently characterized the metal binding and structural features of isoform 1 (TgCEN1), demonstrating that it possesses properties consistent with a role as a Ca2+ sensor and displays a Ca2+-dependent tendency to self-assemble. Herein, we expanded our studies, focusing on the self-association and target binding properties of TgCEN1 by combining biophysical techniques including dynamic light scattering, isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy. We found that the self-assembly process of TgCEN1 depends on different physicochemical factors, including Ca2+ concentration, temperature, and protein concentration, and is mediated by both electrostatic and hydrophobic interactions. The process is completely abolished upon removal of the first 21-residues of the protein and is significantly reduced in the presence of a binding target peptide derived from the human XPC protein (P17-XPC). Titration of P17-XPC to the intact protein and isolated domains showed that TgCEN1 possesses two binding sites with distinct affinities and Ca2+ sensitivity; a high-affinity site in the C-lobe which may be constitutively bound to the peptide and a low-affinity site in the N-lobe which is active only upon Ca2+ stimulus. Overall, our results suggest a specific mechanism of TgCEN1 for Ca2+-modulated target binding and support a N-to-C self-assembly mode, in which the first 21-residues of one molecule likely interact with the C-lobe of the other., (© 2021 The Author(s).)

Centrins are calcium (Ca 2+ )-binding proteins that have been implicated in several regulatory functions. In the protozoan parasite Toxoplasma gondii , the causative agent of toxoplasmosis, three isoforms of centrin have been identified. While increasing information is now available that links the function of centrins with defined parasite biological processes, knowledge is still limited on the metal-binding and structural properties of these proteins. Herein, using biophysical and structural approaches, we explored the Ca 2+ binding abilities and the subsequent effects of Ca 2+ on the structure of a conserved (TgCEN1) and a more divergent (TgCEN2) centrin isoform from T. gondii . Our data showed that TgCEN1 and TgCEN2 possess diverse molecular features, suggesting that they play nonredundant roles in parasite physiology. TgCEN1 binds two Ca 2+ ions with high/medium affinity, while TgCEN2 binds one Ca 2+ with low affinity. TgCEN1 undergoes significant Ca 2+ -dependent conformational changes that expose hydrophobic patches, supporting a role as a Ca 2+ sensor in toxoplasma. In contrast, Ca 2+ binding has a subtle influence on conformational features of TgCEN2 without resulting in hydrophobic exposure, suggesting a different Ca 2+ relay mode for this isoform. Furthermore, TgCEN1 displays a Ca 2+ -dependent ability to self-assemble, while TgCEN2 did not. We discuss our findings in the context of Ca 2+ signaling in toxoplasma.

Background : KDM5 enzymes are H3K4 specific histone demethylases involved in transcriptional regulation and DNA repair. These proteins are overexpressed in different kinds of cancer, including breast, prostate and bladder carcinomas, with positive effects on cancer proliferation and chemoresistance. For these reasons, these enzymes are potential therapeutic targets. Methods : In the present study, we analyzed the effects of three different inhibitors of KDM5 enzymes in MCF-7 breast cancer cells over-expressing one of them, namely KDM5B/JARID1B. In particular we tested H3K4 demethylation (western blot); radio-sensitivity (cytoxicity and clonogenic assays) and damage accumulation (COMET assay and kinetics of H2AX phosphorylation). Results : we show that all three compounds with completely different chemical structures can selectively inhibit KDM5 enzymes and are capable of increasing sensitivity of breast cancer cells to ionizing radiation and radiation-induced damage. Conclusions : These findings confirm the involvement of H3K4 specific demethylases in the response to DNA damage, show a requirement of the catalytic function and suggest new strategies for the therapeutic use of their inhibitors.