RESUMO
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.
Assuntos
Celulose , Oxigenases de Função Mista , Celulose/química , Celulose/metabolismo , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/química , Podospora/enzimologia , Polissacarídeos/química , Polissacarídeos/metabolismo , BiomassaRESUMO
Mutualistic 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.
Assuntos
Clorófitas , Líquens , Filogenia , Simbiose , Líquens/genética , Líquens/microbiologia , Simbiose/genética , Clorófitas/genética , Transferência Genética Horizontal , Evolução Molecular , Evolução Biológica , Transcriptoma , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , GenômicaRESUMO
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 CoAA9A, 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 CoAA9A 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.
Assuntos
Proteínas Fúngicas , Polissacarídeos , Multimerização Proteica , Espalhamento a Baixo Ângulo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Difração de Raios X , Polissacarídeos/metabolismo , Celulose/metabolismoRESUMO
White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H218O2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.
Assuntos
Basidiomycota , Peróxido de Hidrogênio , Polyporaceae , Catalase/metabolismo , Peróxido de Hidrogênio/metabolismo , Madeira/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Fúngicas/metabolismo , Lignina/metabolismo , Basidiomycota/metabolismo , Oxirredução , Celulose 1,4-beta-Celobiosidase/metabolismo , Carboidratos , Metionina/metabolismo , Sulfonas/metabolismoRESUMO
Lytic polysaccharide monooxygenases (LPMOs) have an essential role in global carbon cycle, industrial biomass processing and microbial pathogenicity by catalysing the oxidative cleavage of recalcitrant polysaccharides. Despite initially being considered monooxygenases, experimental and theoretical studies show that LPMOs are essentially peroxygenases, using a single copper ion and H2O2 for C-H bond oxygenation. Here, we examine LPMO catalysis, emphasizing key studies that have shaped our comprehension of their function, and address side and competing reactions that have partially obscured our understanding. Then, we compare this novel copper-peroxygenase reaction with reactions catalysed by haem iron enzymes, highlighting the different chemistries at play. We conclude by addressing some open questions surrounding LPMO catalysis, including the importance of peroxygenase and monooxygenase reactions in biological contexts, how LPMOs modulate copper site reactivity and potential protective mechanisms against oxidative damage.
Assuntos
Peróxido de Hidrogênio , Metaloproteínas , Peróxido de Hidrogênio/química , Cobre/química , Polissacarídeos/química , Oxigenases de Função Mista/química , CatáliseRESUMO
Lytic polysaccharide monooxygenases (LPMOs) can perform oxidative cleavage of glycosidic bonds in carbohydrate polymers (e.g., cellulose, chitin), making them more accessible to hydrolytic enzymes. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. The AA10 LPMOs are active on chitin and/or cellulose and mostly found in bacteria and in some viruses and archaea. Interestingly, AA10-encoding genes are also encountered in some pathogenic fungi of the Ustilaginomycetes class, such as Ustilago maydis, responsible for corn smut disease. Transcriptomic studies have shown the overexpression of the AA10 gene during the infectious cycle of U. maydis. In fact, U. maydis has a unique AA10 gene that codes for a catalytic domain appended with a C-terminal disordered region. To date, there is no public report on fungal AA10 LPMOs. In this study, we successfully produced the catalytic domain of this LPMO (UmAA10_cd) in Pichia pastoris and carried out its biochemical characterization. Our results show that UmAA10_cd oxidatively cleaves α- and ß-chitin with C1 regioselectivity and boosts chitin hydrolysis by a GH18 chitinase from U. maydis (UmGH18A). Using a biologically relevant substrate, we show that UmAA10_cd exhibits enzymatic activity on U. maydis fungal cell wall chitin and promotes its hydrolysis by UmGH18A. These results represent an important step toward the understanding of the role of LPMOs in the fungal cell wall remodeling process during the fungal life cycle.IMPORTANCELytic polysaccharide monooxygenases (LPMOs) have been mainly studied in a biotechnological context for the efficient degradation of recalcitrant polysaccharides. Only recently, alternative roles and paradigms begin to emerge. In this study, we provide evidence that the AA10 LPMO from the phytopathogen Ustilago maydis is active against fungal cell wall chitin. Given that chitin-active LPMOs are commonly found in microbes, it is important to consider fungal cell wall as a potential target for this enigmatic class of enzymes.
Assuntos
Quitina , Polissacarídeos , Quitina/metabolismo , Polissacarídeos/metabolismo , Oxigenases de Função Mista/metabolismo , Celulose/metabolismo , Parede Celular/metabolismoRESUMO
Lytic polysaccharide monooxygenases (LPMOs) are taxonomically widespread copper-enzymes boosting biopolymers conversion (e.g. cellulose, chitin) in Nature. White-rot Polyporales, which are major fungal wood decayers, may possess up to 60 LPMO-encoding genes belonging to the auxiliary activities family 9 (AA9). Yet, the functional relevance of such multiplicity remains to be uncovered. Previous comparative transcriptomic studies of six Polyporales fungi grown on cellulosic substrates had shown the overexpression of numerous AA9-encoding genes, including some holding a C-terminal domain of unknown function ("X282"). Here, after carrying out structural predictions and phylogenetic analyses, we selected and characterized six AA9-X282s with different C-term modularities and atypical features hitherto unreported. Unexpectedly, after screening a large array of conditions, these AA9-X282s showed only weak binding properties to cellulose, and low to no cellulolytic oxidative activity. Strikingly, proteomic analysis revealed the presence of multiple phosphorylated residues at the surface of these AA9-X282s, including a conserved residue next to the copper site. Further analyses focusing on a 9 residues glycine-rich C-term extension suggested that it could hold phosphate-binding properties. Our results question the involvement of these AA9 proteins in the degradation of plant cell wall and open new avenues as to the divergence of function of some AA9 members.
Assuntos
Basidiomycota , Cobre , Filogenia , Cobre/metabolismo , Proteômica , Polissacarídeos/metabolismo , Celulose/metabolismo , Basidiomycota/metabolismo , Fosfatos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
Lytic polysaccharide monooxygenases (LPMOs) belonging to the AA14 family are believed to contribute to the enzymatic degradation of lignocellulosic biomass by specifically acting on xylan in recalcitrant cellulose-xylan complexes. Functional characterization of an AA14 LPMO from Trichoderma reesei, TrAA14A, and a re-evaluation of the properties of the previously described AA14 from Pycnoporus coccineus, PcoAA14A, showed that these proteins have oxidase and peroxidase activities that are common for LPMOs. However, we were not able to detect activity on cellulose-associated xylan or any other tested polysaccharide substrate, meaning that the substrate of these enzymes remains unknown. Next to raising questions regarding the true nature of AA14 LPMOs, the present data illustrate possible pitfalls in the functional characterization of these intriguing enzymes.
Assuntos
Oxigenases de Função Mista , Xilanos , Oxigenases de Função Mista/química , Xilanos/metabolismo , Polissacarídeos/metabolismo , Celulose/metabolismo , OxirredutasesRESUMO
Lytic polysaccharide monooxygenase (LPMO) enzymes have recently shaken up our knowledge of the enzymatic degradation of biopolymers and cellulose in particular. This unique class of metalloenzymes cleaves cellulose and other recalcitrant polysaccharides using an oxidative mechanism. Despite their potential in biomass saccharification and cellulose fibrillation, the detailed mode of action of LPMOs at the surface of cellulose fibers still remains poorly understood and highly challenging to investigate. In this study, we first determined the optimal parameters (temperature, pH, enzyme concentration, and pulp consistency) of LPMO action on the cellulose fibers by analyzing the changes in molar mass distribution of solubilized fibers using high performance size exclusion chromatography (HPSEC). Using an experimental design approach with a fungal LPMO from the AA9 family (PaLPMO9H) and cotton fibers, we revealed a maximum decrease in molar mass at 26.6 °C and pH 5.5, with 1.6% w/w enzyme loading in dilute cellulose dispersions (100 mg of cellulose at 0.5% w/v). These optimal conditions were used to further investigate the effect of PaLPMO9H on the cellulosic fiber structure. Direct visualization of the fiber surface by scanning electron microscopy (SEM) revealed that PaLPMO9H created cracks on the cellulose surface while it attacked tension regions that triggered the rearrangement of cellulose chains. Solid-state NMR indicated that PaLPMO9H increased the lateral fibril dimension and created novel accessible surfaces. This study confirms the LPMO-driven disruption of cellulose fibers and extends our knowledge of the mechanism underlying such modifications. We hypothesize that the oxidative cleavage at the surface of the fibers releases the tension stress with loosening of the fiber structure and peeling of the surface, thereby increasing the accessibility and facilitating fibrillation.
Assuntos
Celulose , Fibra de Algodão , Celulose/química , Polissacarídeos/metabolismo , Oxigenases de Função Mista/química , OxirreduçãoRESUMO
Carbohydrate active enzymes (CAZymes) and their biochemical characterization have been the subject of extensive research over the past ten years due to their importance to carbohydrate metabolism in different biological contexts. For instance, the understanding that 'polysaccharide utilizing loci' (PUL) systems hosted by specific 'carbohydrate degraders' in the intestinal microbiota play key roles in health and disease, such as Crohn's disease, ulcerative colitis or colorectal cancer to name the most well-characterized, has led to an outstanding effort in trying to decipher the molecular mechanisms by which these processes are organized and regulated. The past 10 years has also seen the expansion of CAZymes with auxiliary activities, such as lytic polysaccharide monooxygenases (LPMOs) or even sulfatases, and interest has grown in general about the enzymes needed to remove the numerous decorations and modifications of complex biomass, such as carbohydrate esterases (CE). Today, the characterization of these 'modifying' enzymes allows us to tackle a much more complex biomass, which presents sulfations, methylations, acetylations or interconnections with lignin. This special issue about CAZyme biochemistry covers all these aspects, ranging from implications in disease to environmental and biotechnological impact, with a varied collection of twenty-four review articles providing current biochemical, structural and mechanistic insights into their respective topics.
Assuntos
Metabolismo dos Carboidratos , Carboidratos , Humanos , Polissacarídeos/metabolismoRESUMO
Considering an ever-growing global population, which hit 8 billion people in the fall of 2022, it is essential to find solutions to avoid the competition between human food and animal feed for croplands. Agricultural co-products have become important components of the circular economy with their use in animal feed. Their implementation was made possible by the addition of exogenous enzymes in the diet, especially carbohydrate-active enzymes (CAZymes). In this review, we describe the diversity and versatility of microbial CAZymes targeting non-starch polysaccharides to improve the nutritional potential of diets containing cereals and protein meals. We focused our attention on cellulases, hemicellulases, pectinases which were often found to be crucial in vivo. We also highlight the performance and health benefits brought by the exogenous addition of enzymatic cocktails containing CAZymes in the diets of monogastric animals. Taking the example of the well-studied commercial cocktail Rovabio™, we discuss the evolution, constraints and future challenges faced by feed enzymes suppliers. We hope that this review will promote the use and development of enzyme solutions for industries to sustainably feed humans in the future.
Assuntos
Ração Animal , Polissacarídeos , Animais , Humanos , Polissacarídeos/metabolismo , Grão Comestível/metabolismo , Enzimas , DietaRESUMO
Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment.
Assuntos
Criptococose , Cryptococcus neoformans , Polissacarídeos Fúngicos , Termotolerância , Humanos , Oxigenases de Função Mista/genética , Virulência , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Polissacarídeos/metabolismo , Parede Celular/metabolismoRESUMO
Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose-containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well-conserved first copper-coordination sphere and overall fold, some members of the AA5_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site-directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5_2 enzymes towards galactose-containing saccharides.
Assuntos
Cobre , Galactose , Cobre/metabolismo , Galactose/química , Oxirredutases/metabolismo , Galactose Oxidase/genética , Galactose Oxidase/química , Galactose Oxidase/metabolismo , Oxirredução , Ceruloplasmina , Álcoois , Especificidade por SubstratoRESUMO
Global food security is endangered by fungal phytopathogens causing devastating crop production losses. Many of these pathogens use specialized appressoria cells to puncture plant cuticles. Here, we unveil a pair of alcohol oxidase-peroxidase enzymes to be essential for pathogenicity. Using Colletotrichum orbiculare, we show that the enzyme pair is cosecreted by the fungus early during plant penetration and that single and double mutants have impaired penetration ability. Molecular modeling, biochemical, and biophysical approaches revealed a fine-tuned interplay between these metalloenzymes, which oxidize plant cuticular long-chain alcohols into aldehydes. We show that the enzyme pair is involved in transcriptional regulation of genes necessary for host penetration. The identification of these infection-specific metalloenzymes opens new avenues on the role of wax-derived compounds and the design of oxidase-specific inhibitors for crop protection.
Assuntos
Proteínas Fúngicas , Metaloproteínas , Proteínas Fúngicas/genética , Células Vegetais , Fungos , VirulênciaRESUMO
Filamentous fungi are keystone microorganisms in the regulation of many processes occurring on Earth, such as plant biomass decay and 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 reannotation of this data set 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 ß-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 ß-1,3-glucanase activity, with a preference for ß-1,3-glucans with short ß-1,6 substitutions, and UmAA3_2-A is a dehydrogenase catalyzing the oxidation of ß-1,3- and ß-1,6-gluco-oligosaccharides into the corresponding aldonic acids. Working on model ß-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 coexpressed, 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. IMPORTANCE Filamentous 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 toward 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.
Assuntos
Glicosídeo Hidrolases , Ustilago , Glicosídeo Hidrolases/metabolismo , Zea mays/metabolismo , Oxirredutases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Filogenia , Parede Celular/metabolismo , Fungos/metabolismo , Plantas/metabolismo , Carboidratos , Glucanos/metabolismoRESUMO
Galactose oxidase (GalOx, EC.1.1.3.9) is one of the most extensively studied copper radical oxidases (CROs). The reaction catalyzed by GalOx leads to the oxidation of the C-6 hydroxyl group of galactose and galactosides (including galactosylated polysaccharides and glycoproteins) to the corresponding aldehydes, coupled to the reduction of dioxygen to hydrogen peroxide. Despite more than 60 years of research including mechanistic studies, enzyme engineering and application development, GalOx activity remains primarily monitored by indirect measurement of the co-product hydrogen peroxide. Here, we describe a simple direct method to measure GalOx activity through the identification of galactosylated oxidized products using high-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD). Using galactose and lactose as representative substrates, we were able to separate and detect the C-6 oxidized products, which were confirmed by LC-MS and NMR analyses to exist in their hydrated (geminal-diol) forms. We show that the HPAEC-PAD method is superior to other methods in terms of sensitivity as we could detect down to 0.08 µM of LacOX (eq. 30 µg L-1). We believe the method will prove useful for qualitative detection of galactose oxidase activity in biological samples or for quantitative purposes to analyze enzyme kinetics or to compare enzyme variants in directed evolution programs.
RESUMO
1D 1H NMR spectroscopy has been widely used to monitor enzymatic activity by recording the evolution of the spectra of substrates and/or products, thanks to the linear response of NMR. For complex systems involving the coexistence of multiple compounds (substrate, final product and various intermediates), the identification and quantification can be a more arduous task. Here, we present a simple analytical method for the rapid characterization of reaction mixtures involving enzymatic complexes using Maximum Quantum (MaxQ) NMR, accelerated with the Non-Uniform Sampling (NUS) acquisition procedure. Specifically, this approach enables, in the first analytical step, the counting of the molecules present in the samples. We also show, using two different enzymatic systems, that the implementation of these pulse sequences implies precautions related to the short relaxation times due to the presence of metallo-enzymes or paramagnetic catalysts. Finally, the combination of MaxQ and diffusion experiments, which leads to a 3D chart, greatly improves the resolution and offers an extreme simplification of the spectra while giving valuable indications on the affinity of the enzymes to the different compounds present in the reaction mixture.
Assuntos
Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodosRESUMO
In the last two decades it has become increasingly evident that a large number of proteins adopt either a fully or a partially disordered conformation. Intrinsically disordered proteins are ubiquitous proteins that fulfill essential biological functions while lacking a stable 3D structure. Their conformational heterogeneity is encoded by the amino acid sequence, thereby allowing intrinsically disordered proteins or regions to be recognized based on their sequence properties. The identification of disordered regions facilitates the functional annotation of proteins and is instrumental for delineating boundaries of protein domains amenable to crystallization. This chapter focuses on the methods currently employed for predicting protein disorder and identifying intrinsically disordered binding sites.
Assuntos
Proteínas Intrinsicamente Desordenadas , Sequência de Aminoácidos , Sítios de Ligação , Proteínas Intrinsicamente Desordenadas/química , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
BACKGROUND: Fungal saccharification of lignocellulosic biomass occurs concurrently with the secretion of a diverse collection of proteins, together functioning as a catalytic system to liberate soluble sugars from insoluble composite biomaterials. How different fungi respond to different substrates is of fundamental interest to the developing biomass saccharification industry. Among the cornerstones of fungal enzyme systems are the highly expressed cellulases (endo-ß-glucanases and cellobiohydrolases). Recently, a cyclophellitol-derived activity-based probe (ABP-Cel) was shown to be a highly sensitive tool for the detection and identification of cellulases. RESULTS: Here we show that ABP-Cel enables endo-ß-glucanase profiling in diverse fungal secretomes. In combination with established ABPs for ß-xylanases and ß-D-glucosidases, we collected multiplexed in-gel fluorescence activity-based protein profiles of 240 secretomes collected over ten days from biological replicates of ten different basidiomycete fungi grown on maltose, wheat straw, or aspen pulp. Our results reveal the remarkable dynamics and unique enzyme fingerprints associated with each species substrate combination. Chemical proteomic analysis identifies significant arsenals of cellulases secreted by each fungal species during growth on lignocellulosic biomass. Recombinant production and characterization of a collection of probe-reactive enzymes from GH5, GH10, and GH12 confirm that ABP-Cel shows broad selectivity towards enzymes with endo-ß-glucanase activity. CONCLUSION: Using small-volume samples with minimal sample preparation, the results presented here demonstrate the ready accessibility of sensitive direct evidence for fungal enzyme secretion during early stages of growth on complex lignocellulosic substrates.