Biology subjects

Grisel, S.

Publications and source records attributed to Grisel, S..

6 recordsLinked to original sources

bioRxiv2025

The C-terminal intrinsically disordered region of a fungal LPMO binds copper and displays anti-fungal properties

Lytic Polysaccharide Monooxygenases (LPMOs) are enzymes that play a crucial role in the degradation of complex polysaccharides such as cellulose and chitin. While LPMOs have attracted significant interest for industrial applications to convert biomass into biofuels, emerging evidence suggests alternative functions in fungal plant pathogenesis, microbial diseases and (micro)organism development. The AA14 LPMO family is widely distributed in filamentous fungi but remains enigmatic as its initially-suspected substrate specificity was recently challenged. In this study, we investigated the disordered C-terminal regions (dCTRs), found in more than half of AA14 family members and hypothesized to have functional relevance. Focusing on the Pycnoporus coccineus AA14A LPMO, we used small angle X-ray scattering (SAXS) and circular dichroism to show that its dCTR is highly disordered. It consists of a heavily glycosylated low complexity region followed by a charged C-terminal tail. We uncovered that the PcoAA14A dCTR binds copper ions through histidine residues located in the C-terminal tail. Using electron paramagnetic resonance (EPR), we further demonstrated that dimer formation occurs through an oxidative process involving copper and a redox-sensitive cysteine. Noting similarities between the last residues of the C-terminal tail and antimicrobial peptides, we investigated its potential antimicrobial function. We found that the positively charged region of the C-terminal tail selectively inhibits the growth of basidiomycete fungal spores. These data reveal that the dCTRs appended to AA14 LPMOs are functional regions that must be considered to unveil the role of these atypical LPMOs. Significance StatementLytic polysaccharide monooxygenases (LPMOs) degrade complex polysaccharides and are central to biomass conversion. Their biological relevance is expanding, with emerging roles in fungal development and host interactions. Despite this broad potential, research has largely focused on their catalytic domain. One area that remains poorly understood is the function of their C-terminal region, predicted to be intrinsically disordered (dCTR). The AA14 LPMO family, whose substrate specificity remains enigmatic, is enriched in dCTRs. This study provides the first experimental evidence of the disordered nature of the dCTR of an AA14 LPMO. Using multidisciplinary approaches, we demonstrate that the dCTR binds metals and exhibits antifungal activity. These findings establish dCTRs as functional regions, essential for understanding the biological roles of LPMOs.

biochemistry↗

bioRxiv2025

Functional insights into a peculiar tetra-modular LPMO from the human pathogen Enterobacter cloacae

Enterobacter cloacae is a Gram-negative nosocomial human pathogen that inhabits diverse ecological niches. Its genome encodes a conserved set of putative chitin-active enzymes, including a peculiar lytic polysaccharide monooxygenase (LPMO), termed EcLPMO, which we functionally characterized in this study. EcLPMO is a tetra-modular protein consisting of an auxiliary activity family 10 (AA10) catalytic domain, two central domains of unknown function (DUF-A and DUF-B), and a C-terminal carbohydrate-binding module (CBM73). Functional assays using full-length EcLPMO and its truncated variants demonstrated that the AA10 domain oxidatively cleaves chitin at the C1 position. The CBM73 module enhances chitin binding and promotes synergy with endogenous chitinases. Notably, EcLPMO displayed a particularly strong synergistic effect with the unimodular chitinase EcChiA, leading to up to 14-fold and 60-fold increases in GlcNAc release from - and {beta}-chitin, respectively. Deletion of both DUFs reduced EcLPMO activity. While DUF-A alone and the association of DUF-A and DUF-B showed limited chitin binding, DUF-B alone exhibited no binding, suggesting a distinct role. Unexpectedly, using state-of-the-art structural modelling (AlphaFold3), we observed that the DUF-B domain contains two highly conserved histidines that coordinate the AA10-bound copper, forming a previously unreported inter-domain tetra-histidine copper coordination center. These findings highlight the structural and functional complexity of EcLPMO and suggest that its accessory domains, particularly DUF-B, may contribute to enzyme stability and substrate interaction. We speculate that DUF-B may protect the LPMO active site from oxidative damage, a feature that could prove crucial in its ecological and pathogenic contexts.

biochemistry↗

bioRxiv2024

Functional carbohydrate-active enzymes acquired by horizontal gene transfer from plants in the whitefly Bemisia tabaci

Carbohydrate-active enzymes (CAZymes) involved in the degradation of plant cell walls and/or the assimilation of plant carbohydrates for energy uptake are widely distributed in microorganisms. In contrast, they are less frequent in animals, although there are exceptions, including examples of CAZymes acquired by horizontal gene transfer (HGT) from bacteria or fungi in several of phytophagous arthropods and plant-parasitic nematodes. Although the whitefly Bemisia tabaci is a major agricultural pest, knowledge of HGT-acquired CAZymes in this phloem-feeding insect of the Hemiptera order (subfamily Aleyrodinae) is still lacking. We performed a comprehensive and accurate detection of HGT candidates in B. tabaci and identified 136 HGT events, 14 of which corresponding to CAZymes. The B. tabaci HGT-acquired CAZymes were not only of bacterial or fungal origin, but some were also acquired from plants. Biochemical analysis revealed that members of the glycoside hydrolase families 17 (GH17) and 152 (GH152) acquired from plants are functional beta-glucanases with different substrate specificities, suggesting distinct roles. These two CAZymes are the first characterized GH17 and GH152 glucanases in an animal. We identified a lower number of HGT events in the related Aleyrodinae Trialeurodes vaporariorum, with only three HGT-acquired CAZymes, including a GH152 glucanase, with phylogenetic analysis suggesting a unique HGT event in the ancestor of the Aleyrodinae. Another GH152 CAZyme, most likely independently acquired from plants, was also identified in two plant cell-feeding insects of the Thysanoptera order, highlighting the importance of plant-acquired CAZymes in the biology of piercing-sucking insects. Significance statementCarbohydrate-active enzymes (CAZymes) are crucial for sugar metabolism. Those involved in plant cell wall degradation are usually absent from animal genomes. In this study, we explored CAZyme repertoires in the genomes of several insects: the phloem-feeding whitefly Bemisia tabaci, a major agricultural pest, and the related greenhouse whitefly Trialeurodes vaporariorum, as well as two Thysanoptera species that feed on plant cell contents. We identified several cases of CAZymes acquired from plant via horizontal gene transfer in the genome of these insects. Notably, we showed that two B. tabaci CAZymes of plant origin function as glucanases with distinct substrate specificities, potentially helping the insect to overcome plant defenses. Overall, these findings enhance our understanding of how the ability to feed on plants evolved in insects.

evolutionary biology↗

bioRxiv2022

The maize pathogen Ustilago maydis secretes glycoside hydrolases and carbohydrate oxidases directed towards components of the fungal cell wall

Filamentous fungi are keystone microorganisms in the regulation of many processes occurring on Earth, such as plant biomass decay, pathogenesis as well as symbiotic associations. In many of these processes, fungi secrete carbohydrate-active enzymes (CAZymes) to modify and/or degrade carbohydrates. Ten years ago, while evaluating the potential of a secretome from the maize pathogen Ustilago maydis to supplement lignocellulolytic cocktails, we noticed it contained many unknown or poorly characterized CAZymes. Here, and after re-annotation of this dataset and detailed phylogenetic analyses, we observed that several CAZymes (including glycoside hydrolases and carbohydrate oxidases) are predicted to act on the fungal cell wall (FCW), notably on {beta}-1,3-glucans. We heterologously produced and biochemically characterized two new CAZymes, called UmGH16_1-A and UmAA3_2-A. We show that UmGH16_1-A displays {beta}-1,3-glucanase activity, with a preference for {beta}-1,3-glucans with short {beta}-1,6 substitutions, and UmAA3_2-A is a dehydrogenase catalyzing the oxidation of {beta}-1,3- and {beta}-1,6-gluco-oligosaccharides into the corresponding aldonic acids. Working on model {beta}-1,3-glucans, we show that the linear oligosaccharide products released by UmGH16_1-A are further oxidized by UmAA3_2-A, bringing to light a putative biocatalytic cascade. Interestingly, analysis of available transcriptomics data indicates that both UmGH16_1-A and UmAA3_2-A are co-expressed, only during early stages of U. maydis infection cycle. Altogether, our results suggest that both enzymes are connected and that additional accessory activities still need to be uncovered to fully understand the biocatalytic cascade at play and its physiological role. ImportanceFilamentous fungi play a central regulatory role on Earth, notably in the global carbon cycle. Regardless of their lifestyle, filamentous fungi need to remodel their own cell wall (mostly composed of polysaccharides) to grow and proliferate. To do so, they must secrete a large arsenal of enzymes, most notably carbohydrate-active enzymes (CAZymes). However, research on fungal CAZymes over past decades has mainly focused on finding efficient plant biomass conversion processes while CAZymes directed at the fungus itself have remained little explored. In the present study, using the maize pathogen Ustilago maydis as model, we set off to evaluate the prevalence of CAZymes directed towards the fungal cell wall during growth of the fungus on plant biomass and characterized two new CAZymes active on fungal cell wall components. Our results suggest the existence of a biocatalytic cascade that remains to be fully understood.

biochemistry↗

bioRxiv2021

Time-resolved secretome analysis of three Colletotrichum species identifies copper radical alcohol oxidases for the production of fatty aldehydes

Copper Radical Alcohol Oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens are attractive for the production of fragrant fatty aldehydes. To investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time-course analyses of the secretomes of three Colletotrichum species (C. graminicola, C. tabacum and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29-52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11-AA13, AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CRO-AlcOx were expressed in Pichia pastoris and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C6 and C8 alcohols into their corresponding fragrant aldehydes. IMPORTANCEIn the context of the industrial shift toward greener processes, the biocatalytic production of aldehydes is of utmost interest owing to their importance as intermediates in preparative chemistry and for their use as flavors and fragrances ingredients. In the search for new biocatalysts, CRO-AlcOx have the potential to become platform enzymes for the oxidation of alcohols to aldehydes. The use of crude fungal secretomes is often seen has an appealing approach by industries since alleviating various costs pertaining to biocatalysts production. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored. This study showed that Colletotrichum species can secrete a broad diversity of copper-containing enzymes, but only little amount of CRO-AlcOx. Thus, recombinant expression remains the most promising approach. The potential of CRO-AlcOx as biocatalyst for flavor and fragrance applications was confirmed through the production of two new enzymes with activity on fatty alcohols.

biochemistry↗

bioRxiv2018

Integrative visual omics of the white-rot fungus Polyporus brumalis exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass.

White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found the fungus possessed an unexpectedly large set of genes coding for enzymes related to lignin degradation, and that these were highly expressed and massively secreted under solid-state fermentation conditions. The examination of interrelated multi-omics patterns revealed the coordinated regulation of lignin-active peroxidases and H2O2-generating enzymes along with the activation of cellular mechanisms for detoxification, which combined to result in the efficient lignin breakdown by the fungus.\n\nImportancePlant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e. agriculture and forestry by-products) are major obstacles for biomass conversions. From a biotechnological aspect, these compounds could be a potential source of aromatic platform molecules for bio-based polymers. Here we describe the extraordinary ability of Polyporus brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide. We observed unusual expansions of gene families coding for; 1) Class II peroxidases involved in lignin degradation; and 2) GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity. Our findings suggested the fungus massively mobilizes this oxidative machinery during growth on wheat straw. Overall, we identified sets of co-regulated enzymes, which could potentially augment the efficiency of biotechnological plant biomass conversions.

bioengineering↗