Auxiliary activity family 7 (AA7) oxidoreductases are fungal flavoenzymes that catalyze the C1 oxidation of diverse oligosaccharides coupled to the reduction of molecular oxygen (oxidase activity) or organic molecules (dehydrogenase activity). These enzymes are predominantly derived from Ascomycota, with a smaller, less-explored fraction from Basidiomycota and Oomycota plant pathogens. Moreover, AA7 members are promising biocatalysts for the selective oxidation of carbohydrates, which is an enduring challenge in catalysis. However, the sequence space of AA7 enzymes remains largely uncharted, particularly within basidiomycota members, and the molecular determinants shaping substrate selectivity remain ill-defined. Focusing on the basidiomycete maize pathogen Ustilago maydis, we explored the boundaries of the AA7 sequence space by identifying and characterizing two enzymes, UmAA7A and UmAA7B, that are divergent from hitherto described members. We found that UmAA7s oxidize both chitooligosaccharides (CHOS) and their deacetylated forms (dCHOS) in an acetylation-site-dependent manner. The X-ray crystal structure of UmAA7A, sequence, and structural comparisons with a model of UmAA7B and other known CHOS-active AA7s, combined with docking analyses on CHOS and dCHOS, revealed specific active-site residues behind this unprecedented substrate specificity. Moreover, a previously not reported combination of a bicovalently tethered FAD and an atypical arrangement of residues at re-side of the FAD cofactor was associated with a mainly dehydrogenase activity profile, contrasting the majority of oxidases in AA7. Altogether, this work reveals the enzymatic oxidation of dCHOS, extending the substrate scope of AA7s and laying the biochemical foundation for uncovering their biological functions during plant infection.

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. Moreover, more than half of AA14 family members display a disordered C-terminal region (dCTR), hypothesized to have functional relevance. In this study, we functionally characterized the Pycnoporus coccineus AA14A LPMO that harbors a dCTR. 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 showed that an oxidative process involving copper and a redox-sensitive cysteine triggers enzyme dimerization. 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.

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 Ec LPMO, which we functionally characterized in this study. Ec LPMO 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 Ec LPMO 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, Ec LPMO displayed a particularly strong synergistic effect with the unimodular chitinase Ec ChiA, leading to up to 14-fold and 60-fold increases in GlcNAc release from α- and β-chitin, respectively. Deletion of both DUFs reduced Ec LPMO 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 Ec LPMO 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. ### Competing Interest Statement The authors have declared no competing interest. Department of Biotechnology, https://ror.org/03tjsyq23, DBT-SAHAJ/BUILDER, (BT/INF/22/SP41176/2020), BT/PR42225/BCE/8/1586/2021 Department of Science and Technology, https://ror.org/0101xrq71, Funds for Infrastructure in Science and Technology (FIST), Level III to the Department of Plant Sciences, UoH Science and Engineering Research Board, https://ror.org/03ffdsr55, CRG/2019/006426 UoH-Institution of Eminence, RC1-20-020 INRAE, EvoFun project PAF_02

Carbohydrate-active enzymes 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 carbohydrate-active enzymes acquired by horizontal gene transfer 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 horizontal gene transfer-acquired carbohydrate-active enzymes in this phloem-feeding insect of the Hemiptera order (subfamily Aleyrodinae) is still lacking. We performed a comprehensive and accurate detection of horizontal gene transfer candidates in B. tabaci and identified 136 horizontal gene transfer events, 14 of which corresponding to carbohydrate-active enzymes. The B. tabaci horizontal gene transfer-acquired carbohydrate-active enzymes 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 and 152 acquired from plants are functional beta-glucanases with different substrate specificities, suggesting distinct roles. These two carbohydrate-active enzymes are the first characterized glycoside hydrolase families 17 and 152 glucanases in an animal. We identified a lower number of horizontal gene transfer events in the related Aleyrodinae Trialeurodes vaporariorum, with only three horizontal gene transfer-acquired carbohydrate-active enzymes, including a glycoside hydrolase family 152 glucanase, with phylogenetic analysis suggesting a unique horizontal gene transfer event in the ancestor of the Aleyrodinae. Another glycoside hydrolase family 152 carbohydrate-active enzyme, 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 carbohydrate-active enzymes in the biology of piercing-sucking insects.

Phytophthora phytopathogens from Oomycota cause devastating crop losses and threaten food security. However, Phytophthora secreted proteins that interact with plant-hosts remain underexplored. Here, auxiliary activity family 7 (AA7) enzymes from Ascomycota and Oomycota phytopathogens were shown to oxidise pectin-derived galacturonic acid and/or oligogalacturonides (OGs). Unique mono-cysteinyl-FAD oxidases with positively-charged active sites, suited to oxidise OGs, were discovered in Phytophthora sojae. The P. sojae OG oxidase genes, prevalent in this genus, were co-transcribed with pectin-degradation counterparts during early infection of soybean. Single OG oxidase knockouts significantly decreased P. sojae biomass in planta, potentially linking OG oxidases to virulence. We propose that oxidation by AA7 enzymes impairs the elicitor activity of OGs, potentially contributing to stealth Oomycota infection. Oxidation of OGs unravels a previously unknown microbial mechanism that contributes to evade plant immune-response against pathogens. Our findings highlight a unique oxidase architecture and hitherto unexplored targets for bioprotection from major plant pathogens.

(R)-Citronellal is one of the key chiral intermediates in the synthesis of the isomer (-)-menthol, one of the most commercialised terpenoid flavours worldwide. Enzymatic approaches could represent a less energy-demanding alternative for its synthesis, such as a previously reported bienzymatic cascade starting from inexpensive, commercially available geraniol. A copper radical oxidase (CgrAlcOx) followed by a flavin-dependent ene reductase (OYE2) were used to obtain (R)-citronellal. Here, we used a metal-affinity immobilisation strategy on the His-tagged enzymes for the cascade and studied enzyme recovery and reusability as well as increased solvent tolerance. After screening a panel of resins for enzyme immobilisation and water-immiscible co-solvents, we successfully obtained 95% conversion to (R)-citronellal with 96.9% enantiomeric excess (ee) in a concurrent cascade after 7 h of reaction time, starting from 10 mM of geraniol.

The breakdown of cellulose is one of the most important reactions in nature1,2 and is central to biomass conversion to fuels and chemicals3. However, the microfibrillar organization of cellulose and its complex interactions with other components of the plant cell wall poses a major challenge for enzymatic conversion4. Here, by mining the metagenomic 'dark matter' (unclassified DNA with unknown function) of a microbial community specialized in lignocellulose degradation, we discovered a metalloenzyme that oxidatively cleaves cellulose. This metalloenzyme acts on cellulose through an exo-type mechanism with C1 regioselectivity, resulting exclusively in cellobionic acid as a product. The crystal structure reveals a catalytic copper buried in a compact jelly-roll scaffold that features a flattened cellulose binding site. This metalloenzyme exhibits a homodimeric configuration that enables in situ hydrogen peroxide generation by one subunit while the other is productively interacting with cellulose. The secretome of an engineered strain of the fungus Trichoderma reesei expressing this metalloenzyme boosted the glucose release from pretreated lignocellulosic biomass under industrially relevant conditions, demonstrating its biotechnological potential. This discovery modifies the current understanding of bacterial redox enzymatic systems devoted to overcoming biomass recalcitrance5-7. Furthermore, it enables the conversion of agro-industrial residues into value-added bioproducts, thereby contributing to the transition to a sustainable and bio-based economy.

Pectin is a complex plant heteropolysaccharide whose structure and function differ depending on its source. In animal feed, breaking down pectin is essential, as its presence increases feed viscosity and reduces nutrient absorption. Soybean meal, a protein-rich poultry feed ingredient, contains significant amounts of pectin, the structure of which remains unclear. Consequently, the enzyme activities required to degrade soybean meal pectin and how they interact are still open questions. In this study, we produced 15 recombinant fungal carbohydrate-active enzymes (CAZymes) identified from fungal secretomes acting on pectin. After observing that these enzymes were not active on soybean meal pectin when used alone, we developed a semiminiaturized method to evaluate their effect as multi-activity cocktails. We designed and tested 12 enzyme pools, containing up to 15 different CAZymes, using several hydrolysis markers. Thanks to our multiactivity enzymatic approach combined with a Pearson correlation matrix, we identified 10 fungal CAZymes efficient on soybean meal pectin, 9 of which originate from Talaromyces versatilis. Based on enzyme specificity and linkage analysis, we propose a structural model for soybean meal pectin. Our findings underscore the importance of combining CAZymes to improve the degradation of agricultural co-products.

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 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. EcLPMO exhibited synergy with the unimodular chitinase EcChiA, resulting in up to 14-fold and 60-fold increase in GlcNAc release from α- and β-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. Using 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.

Basidiomycete fungi are the main decomposers of dead wood with an impact on the global carbon cycle. Their degradative mechanisms have been well-studied under aerobic conditions. Here, we study their activity in oxygen-depleted environments. We use metaproteomics in a field study to identify active wood-decomposing fungi and their enzymes at different depths from the wood surface, including in oxygen-depleted conditions. In vitro, we observe that the brown-rot fungus Fomitopsis pinicola can grow on wood in complete anoxia. Using 13C solid-state NMR, we demonstrate the degradation of plant cell-wall polysaccharides and fungal growth in the absence of oxygen. Proteomic analyses reveal that F. pinicola switches from a Fenton chemistry-based process under aerobic conditions to the secretion of plant cell wall-active enzymes in anoxia. Our finding that wood decay fungi can thrive in complete anoxia provides a deeper understanding of lignocellulose degradation mechanisms in nature and raises opportunities for the development of bio-inspired anaerobic processes.

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 Pco AA14A 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 Statement Lytic 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. ### Competing Interest Statement The authors have declared no competing interest.

Lytic polysaccharide monooxygenase (LPMO)-catalyzed oxidative processes play a major role in natural biomass conversion. Despite their oxidative cleavage at the surface of polysaccharides, understanding of their mode of action, and the impact of structural patterns of the cellulose fiber on LPMO activity is still not fully understood. In this work, we investigated the action of two different LPMOs from Podospora anserina on celluloses showing different structural patterns. For this purpose, we prepared cellulose II and cellulose III allomorphs from cellulose I cotton linters, as well as amorphous cellulose. LPMO action was monitored in terms of surface morphology, molar mass changes and monosaccharide profile. Both PaLPMO9E and PaLPMO9H were active on the different cellulose allomorphs (I, II and III), and on amorphous cellulose (PASC) whereas they displayed a different behavior, with a higher molar mass decrease observed for cellulose I. Overall, the pretreatment with LPMO enzymes clearly increased the accessibility of all types of cellulose, which was quantified by the higher carboxylate content after carboxymethylation reaction on LPMO-pretreated celluloses. This work gives more insight into the action of LPMOs as a tool for deconstructing lignocellulosic biomass to obtain new bio-based building blocks.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant polysaccharides such as cellulose. Several studies have reported LPMO action in synergy with other carbohydrate-active enzymes (CAZymes) for the degradation of lignocellulosic biomass but direct LPMO action at the plant tissue level remains challenging to investigate. Here, we have developed a MALDI-MS imaging workflow to detect oxidised oligosaccharides released by a cellulose-active LPMO at cellular level on maize tissues. Using this workflow, we imaged LPMO action and gained insight into the spatial variation and relative abundance of oxidised and non-oxidised oligosaccharides. We reveal a targeted action of the LPMO related to the composition and organisation of plant cell walls.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called Co AA9A, up-regulated during plant infection by Colletotrichum orbiculare , the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates Co AA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.

The worldwide accumulation of plastic waste in the environment, along with its lifespan of hundreds of years, represents a serious threat to ecosystems. Enzymatic recycling of plastic waste offers a promising solution, but the high chemical inertness and hydrophobicity of plastics pose several challenges to enzymes. In nature, lytic polysaccharide monooxygenases (LPMOs) can act at the surface of recalcitrant biopolymers, taking advantage of their solvent-exposed active sites and appended carbohydrate-binding modules (CBMs). LPMOs can disrupt the densely packed chains of polysaccharides (e.g., cellulose) by the oxidation of C-H bonds. Given the similarities between these natural and artificial polymers, we aimed here at promoting plastic-binding properties to LPMOs, by swapping their CBM with three natural, surface-active accessory modules displaying different amphipathic properties. The polymer binding capacity of the resulting LPMO chimeras was assessed on a library of synthetic polymers, including polyester, polyamide, and polyolefin substrates. We demonstrated that the plastic binding properties of these engineered LPMOs are polymer-dependent and can be tuned by playing on the nature of the accessory module and reaction conditions. Remarkably, we gained full binding for some chimera LPMOs with striking results for polyhydroxyalkanoates (PHA). In the long term perspective of harnessing the unique copper chemistry of LPMOs to degrade plastics, we also provided the first evidence of LPMO-dependent modification of the PHA polymer, as supported by enzyme assays, gel permeation chromatography, and scanning electron microscopy. Altogether, our study provides the first roadmap for engineering plastic-binding ability in LPMOs, constituting a crucial first step on the evolutionary path toward efficient interfacial catalysis of plastic-active enzymes.

Plant biomass can be converted to bioproducts using fungal enzymatic cocktails that have been improved by the implementation of lytic polysaccharide monooxygenases (LPMOs) known to boost cellulases. However, due to the complexity of their oxidative catalysis, it is still difficult to properly control and sustain LPMO activity. Here, we investigated whether the implementation of the natural enzymatic partner of LPMOs, i.e., cellobiose dehydrogenase (CDH), could be a promising solution. To this end, we reconstituted a minimal cocktail using the main cellulases from Trichoderma reesei and evaluated the impact of the addition of two LPMOs and one CDH on the conversion of wheat straw, miscanthus, pine, and poplar. Surprisingly, while the addition of LPMOs or LPMO/CDH showed little to no increase in glucose equivalent yields, the sole addition of CDH caused an increase of up to 27% (on miscanthus). Interestingly, CDH supplementation allowed tuning of the stream of degradation products toward cellobionic acid while maintaining equivalent yields or increasing the overall conversion yields in a biomass-dependent manner. Our study shows that extracting reasonable amounts of high-value-added oxidized sugars from industrial biomass is feasible, thereby opening new perspectives for the use of CDH in industry.

AbstractMutualistic symbioses have contributed to major transitions in the evolution of life. Here, we investigate the evolutionary history and the molecular innovations at the origin of lichens, which are a symbiosis established between fungi and green algae or cyanobacteria. We de novo sequence the genomes or transcriptomes of 12 lichen algal symbiont (LAS) and closely related non-symbiotic algae (NSA) to improve the genomic coverage of Chlorophyte algae. We then perform ancestral state reconstruction and comparative phylogenomics. We identify at least three independent gains of the ability to engage in the lichen symbiosis, one in Trebouxiophyceae and two in Ulvophyceae, confirming the convergent evolution of the lichen symbioses. A carbohydrate-active enzyme from the glycoside hydrolase 8 (GH8) family was identified as a top candidate for the molecular-mechanism underlying lichen symbiosis in Trebouxiophyceae. This GH8 was acquired in lichenizing Trebouxiophyceae by horizontal gene transfer, concomitantly with the ability to associate with lichens fungal symbionts (LFS) and is able to degrade polysaccharides found in the cell wall of LFS. These findings indicate that a combination of gene family expansion and horizontal gene transfer provided the basis for lichenization to evolve in chlorophyte algae.

Sesquiterpene cyclases (STC) catalyse the cyclization of the C15 molecule farnesyl diphosphate into a vast variety of mono- or polycyclic hydrocarbons and, for a few enzymes, oxygenated structures, with diverse stereogenic centres. The huge diversity in sesquiterpene skeleton structures in nature is primarily the result of the type of cyclization driven by the STC. Despite the phenomenal impact of fungal sesquiterpenes on the ecology of fungi and their potentials for applications, the fungal sesquiterpenome is largely untapped. The identification of fungal STC is generally based on protein sequence similarity with characterized enzymes. This approach has improved our knowledge on STC in a few fungal species, but it has limited success for the discovery of distant sequences. Besides, the tools based on secondary metabolite biosynthesis gene clusters have shown poor performance for terpene cyclases. Here, we used four sets of sequences of fungal STC that catalyse four types of cyclization, and specific amino acid motives to identify phylogenetically related sequences in the genomes of basidiomycetes fungi from the order Polyporales. We validated that four STC genes newly identified from the genome sequence of Leiotrametes menziesii, each classified in a different phylogenetic clade, catalysed a predicted cyclization of farnesyl diphosphate. We built HMM models and searched STC genes in 656 fungal genomes genomes. We identified 5605 STC genes, which were classified in one of the four clades and had a predicted cyclization mechanism. We noticed that the HMM models were more accurate for the prediction of the type of cyclization catalysed by basidiomycete STC than for ascomycete STC.