High Substrate Specificity Research Articles

Analysis of ligand binding and resulting conformational changes in pyrophosphatase NUDT9.

Nudix hydrolase 9 (NUDT9) is a member of the nucleoside linked to another moiety X (NUDIX) protein superfamily, which hydrolyses a broad spectrum of organic pyrophosphates from metabolic processes. ADP‐ribose (ADPR) has been the only known endogenous substrate accepted by NUDT9 so far. The Ca2+‐permeable transient receptor potential melastatin subfamily 2 (TRPM2) channel contains a homologous NUDT9‐homology (NUDT9H) domain and is activated by ADPR. Sustained Ca2+ influx via ADPR‐activated TRPM2 triggers apoptotic mechanisms. Thus, a precise regulation of cellular ADPR levels by NUDT9 is essential. A detailed characterization of the enzyme‐substrate interaction would help to understand the high substrate specificity of NUDT9. Here, we analysed ligand binding to NUDT9 using a variety of biophysical techniques. We identified 2′‐deoxy‐ADPR as an additional substrate for NUDT9. Similar enzyme kinetics and binding affinities were determined for the two ligands. The high‐affinity binding was preserved in NUDT9 containing the mutated NUDIX box derived from the human NUDT9H domain. NMR spectroscopy indicated that ADPR and 2′‐deoxy‐ADPR bind to the same binding site of NUDT9. Backbone resonance assignment and subsequent molecular docking allowed further characterization of the binding pocket. Substantial conformational changes of NUDT9 upon ligand binding were observed which might allow for the development of NUDT9‐based ADPR fluorescence resonance energy transfer sensors that may help with the analysis of ADPR signalling processes in cells in the future.

Acid Stable Yeast Cell-Associated Tannase with High Capability in Gallated Catechin Biotransformation.

Previously, nine tannin-tolerant and tannase-producing yeasts were isolated from Miang; all produced cell-associated tannase (CAT) during growth in tannin substrate. Among which, only CAT from Sporidiobolus ruineniae showed better stability than its purified form. Yet, it is of particular interest to directly characterize CATs from the latter yeasts. In this study, four CATs from yeasts, namely Cyberlindnera rhodanensis A22.3, Candida sp. A39.3, Debaryomyces hansenii A45.1, and Cy. rhodanensis A45.3 were characterized. The results indicate that all CATs were produced within the same production yield (11 mU/mL). Most CATs exhibited similar pH and temperature optima and stabilities, except for CAT from Cy. rhodanensis A22.3. This CAT was assigned as acid-stable tannase due to its unusual optimum pH of 2.0 with pH stability and half-life thermostability in the range of pH 2.0–4.0, and 70 °C, respectively. All CATs demonstrated high substrate specificity toward epigallocatechin gallate and epicatechin gallate, thus forming epigallocatechin and epicatechin, respectively. Moreover, they showed operational stability to repeated use for up to five cycles without loss of the initial activity. Therefore, CATs from these yeasts could be useful for the extraction and biotransformation of tea catechins and related applications.

Identification and Functional Characterization of Genes Encoding Phenylacetaldehyde Reductases That Catalyze the Last Step in the Biosynthesis of Hydroxytyrosol in Olive.

Hydroxytyrosol derivatives are the most important phenolic components in virgin olive oil due to their well-demonstrated biological activities. In this regard, two phenyl acetaldehyde reductase genes, OePAR1.1 and OePAR1.2, involved in hydroxytyrosol synthesis, have been identified from an olive transcriptome. Both genes were synthesized and expressed in Escherichia coli, and their encoded proteins were purified. The recombinant enzymes display high substrate specificity for 2,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) to form hydroxytyrosol. The reaction catalyzed by OePAR constitutes the second, and last, biochemical step in the formation of hydroxytyrosol from the amino acid L-3,4-dihydroxyphenylalanine (L-DOPA) in olive. OePAR1.1 and OePAR1.2 enzymes exhibit high thermal stability, similar pH optima (pH 6.5), and high affinity for 3,4-DHPAA (apparent Km 0.6 and 0.8 µmol min−1 mg−1, respectively). However, OePAR1.2 exhibited higher specific activity and higher expression levels in all the olive cultivars under study. The expression analyses indicate that both OePAR1.1 and OePAR1.2 genes are temporally regulated in a cultivar-dependent manner. The information provided here could be of interest for olive breeding programs searching for new olive genotypes with the capacity to produce oils with higher levels of hydroxytyrosol derivatives.

Structural basis for substrate specificity of the peroxisomal acyl-CoA hydrolase MpaH' involved in mycophenolic acid biosynthesis.

Mycophenolic acid (MPA) is a fungal natural product and first-line immunosuppressive drug for organ transplantations and autoimmune diseases. In the compartmentalized biosynthesis of MPA, the acyl-coenzyme A (CoA) hydrolase MpaH' located in peroxisomes catalyzes the highly specific hydrolysis of MPA-CoA to produce the final product MPA. The strict substrate specificity of MpaH' not only averts undesired hydrolysis of various cellular acyl-CoAs, but also prevents MPA-CoA from further peroxisomal β-oxidation catabolism. To elucidate the structural basis for this important property, in this study, we solve the crystal structures of the substrate-free form of MpaH' and the MpaH'S139A mutant in complex with the product MPA. The MpaH' structure reveals a canonical α/β-hydrolase fold with an unusually large cap domain and a rare location of the acidic residue D163 of catalytic triad after strand β6. MpaH' also forms an atypical dimer with the unique C-terminal helices α13 and α14 arming the cap domain of the other protomer and indirectly participating in the substrate binding. With these characteristics, we propose that MpaH' and its homologs form a new subfamily of α/β hydrolase fold protein. The crystal structure of MpaH'S139A /MPA complex and the modeled structure of MpaH'/MPA-CoA, together with the structure-guided mutagenesis analysis and isothermal titration calorimetry (ITC) measurements, provide important mechanistic insights into the high substrate specificity of MpaH'.

Secondary Alcohol Dehydrogenases from Thermoanaerobacter pseudoethanolicus and Thermoanaerobacter brockii as Robust Catalysts.

Alcohol dehydrogenases (ADHs) are an important type of enzyme that have significant applications as biocatalysts. Secondary ADHs from Thermoanaerobacter pseudoethanolicus (TeSADH) and Thermoanaerobacter brockii (TbSADH) are well-known as robust catalysts. However, like most other ADHs, these enzymes suffer from their high substrate specificities (i. e., limited substrate scope), which to some extent restricts their use as biocatalysts. This minireview discusses recent efforts to expand the substrate scope and tune the enantioselectivity of TeSADH and TbSADH by using site-directed mutagenesis and directed evolution. Various examples of asymmetric synthesis of optically active alcohols using both enzymes are highlighted. Moreover, the unique thermal stability and organic solvent tolerance of these enzymes is illustrated by their concurrent inclusion with other interesting reactions to synthesize optically active alcohols and amines. For instance, TeSADH has been used in quantitative non-stereoselective oxidation of alcohols to deracemize alcohols via cyclic deracemization and in the racemization of enantiopure alcohols to accomplish a bienzymatic dynamic kinetic resolution.

Discovery of a New, Recurrent Enzyme in Bacterial Phosphonate Degradation: (R)-1-Hydroxy-2-aminoethylphosphonate Ammonia-lyase.

Phosphonates represent an important source of bioavailable phosphorus in certain environments. Accordingly, many microorganisms (particularly marine bacteria) possess catabolic pathways to degrade these molecules. One example is the widespread hydrolytic route for the breakdown of 2-aminoethylphosphonate (AEP, the most common biogenic phosphonate). In this pathway, the aminotransferase PhnW initially converts AEP into phosphonoacetaldehyde (PAA), which is then cleaved by the hydrolase PhnX to yield acetaldehyde and phosphate. This work focuses on a pyridoxal 5′-phosphate-dependent enzyme that is encoded in >13% of the bacterial gene clusters containing the phnW–phnX combination. This enzyme (which we termed PbfA) is annotated as a transaminase, but there is no obvious need for an additional transamination reaction in the established AEP degradation pathway. We report here that PbfA from the marine bacterium Vibrio splendidus catalyzes an elimination reaction on the naturally occurring compound (R)-1-hydroxy-2-aminoethylphosphonate (R-HAEP). The reaction releases ammonia and generates PAA, which can be then hydrolyzed by PhnX. In contrast, PbfA is not active toward the S enantiomer of HAEP or other HAEP-related compounds such as ethanolamine and d,l-isoserine, indicating a very high substrate specificity. We also show that R-HAEP (despite being structurally similar to AEP) is not processed efficiently by the PhnW–PhnX couple in the absence of PbfA. In summary, the reaction catalyzed by PbfA serves to funnel R-HAEP into the hydrolytic pathway for AEP degradation, expanding the scope and the usefulness of the pathway itself.

Reconciling conformational heterogeneity and substrate recognition in cytochrome P450

Cytochrome P450, the ubiquitous metalloenzyme involved in detoxification of foreign components, has remained one of the most popular systems for substrate-recognition process. However, despite being known for its high substrate specificity, the mechanistic basis of substrate-binding by archetypal system cytochrome P450cam has remained at odds with the contrasting reports of multiple diverse crystallographic structures of its substrate-free form. Here, we address this issue by elucidating the probability of mutual dynamical transition to the other crystallographic pose of cytochrome P450cam and vice versa via unbiased all-atom computer simulation. A robust Markov state model, constructed using adaptively sampled 84-μs-long molecular dynamics simulation trajectories, maps the broad and heterogenous P450cam conformational landscape into five key substates. In particular, the Markov state model identifies an intermediate-assisted dynamic equilibrium between a pair of conformations of P450cam, in which the substrate-recognition sites remain “closed” and “open,” respectively. However, the estimate of a significantly higher stationary population of closed conformation, coupled with faster rate of open → closed transition than its reverse process, dictates that the net conformational equilibrium would be swayed in favor of “closed” conformation. Together, the investigation quantitatively infers that although a potential substrate of cytochrome P450cam would, in principle, explore a diverse array of conformations of substrate-free protein, it would mostly encounter a “closed” or solvent-occluded conformation and hence would follow an induced-fit-based recognition process. Overall, the work reconciles multiple precedent crystallographic, spectroscopic investigations and establishes how a statistical elucidation of conformational heterogeneity in protein would provide crucial insights in the mechanism of potential substrate-recognition process.

Structure-guided protein engineering of human cathepsin L for efficient collagenolytic activity.

Engineering precise substrate specificity of proteases advances the potential to use them in biotechnological and therapeutic applications. Collagen degradation, a physiological process mediated by collagenases, is an integral part of extracellular matrix remodeling and when uncontrolled, implicated in different pathological conditions. Lysosomal cathepsin-K cleaves triple helical collagen fiber, whereas cathepsin-L cannot do so. In this study, we have imparted collagenolytic property to cathepsin-L, by systematically engineering proline-specificity and glycosaminoglycans (GAG)-binding surface in the protease. The proline-specific mutant shows high specificity for prolyl-peptidic substrate but is incapable of cleaving collagen. Engineering a GAG-binding surface on the proline-specific mutant enabled it to degrade type-I collagen in the presence of chondroitin-4-sulfate (C4-S). We also present the crystal structures of proline-specific (1.4Å) and collagen-specific (1.8Å) mutants. Finally docking studies with prolyl-peptidic substrate (Ala-Gly-Pro-Arg-Ala) at the active site and a C4-S molecule at the GAG-binding site enable us to identify key structural features responsible for collagenolytic activity of cysteine cathepsins.

Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus.

Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent. In this study, gs-MDH was engineered to increase its catalytic activity at low temperatures. Based on sequential and structural comparison with lactate dehydrogenase from G. stearothermophilus, we selected G218 as a mutation site to increase the loop flexibility pivotal for MDH catalysis. The G218 mutants showed significantly higher specific activities than the wild type at low temperatures and maintained thermal stability. The crystal structure of the G218Y mutant, which had the highest catalytic efficiency among all the G218 mutants, suggested that the flexibility of the mobile loop was successfully increased by the bulky side chain. Therefore, this study demonstrated the low-temperature adaptation of MDH by facilitating conformational changes during catalysis.

Regulatory inter-domain interactions influence Hsp70 recruitment to the DnaJB8 chaperone

The Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.

Dual inhibition of SARS-CoV-2 and human rhinovirus with protease inhibitors in clinical development

The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered a major target for the discovery of direct antiviral agents. We previously reported the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay (Gurard-Levin et al., 2020). The assay was further improved by adding the rhinovirus HRV3C protease to the same well as the SARS-CoV-2 3CLpro enzyme. High substrate specificity for each enzyme allowed the proteases to be combined in a single assay reaction without interfering with their individual activities. This novel duplex assay was used to profile a diverse set of reference protease inhibitors. The protease inhibitors were grouped into three categories based on their relative potency against 3CLpro and HRV3C including those that are: equipotent against 3CLpro and HRV3C (GC376 and calpain inhibitor II), selective for 3CLpro (PF-00835231, calpain inhibitor XII, boceprevir), and selective for HRV3C (rupintrivir). Structural analysis showed that the combination of minimal interactions, conformational flexibility, and limited bulk allows GC376 and calpain inhibitor II to potently inhibit both enzymes. In contrast, bulkier compounds interacting more tightly with pockets P2, P3, and P4 due to optimization for a specific target display a more selective inhibition profile. Consistently, the most selective viral protease inhibitors were relatively weak inhibitors of human cathepsin L. Taken together, these results can guide the design of cysteine protease inhibitors that are either virus-specific or retain a broad antiviral spectrum against coronaviruses and rhinoviruses.

Microbial therapeutic enzymes: A promising area of biopharmaceuticals

• Microbial enzymes are prospective biopharmaceuticals. • They are potential therapeutic agents being economical, consistent, convenient in isolation and genetic manipulation. • They have diverse applications as enzybiotics, digestive-aids, anti-inflammatory, fibrinolytic and anti-cancer agents. • Exploring their curative potential and optimization of pharmacokinetics using suitable nanocarriers can revolutionize healthcare. Biopharmaceuticals is a rapidly emerging field that explores and employs biological organisms and their products for therapeutic applications. Enzymes are remarkable biocatalysts that accelerate the rate of biochemical reactions manifold. They possess several extraordinary features like immense catalytic potential, high substrate specificity as well as pH and temperature optima. Due to these extraordinary attributes, they find diverse clinical applications. They are being increasingly employed for the treatment of a wide spectrum of diseases, either alone or in combination with other therapies. Functional enzymes can be isolated from plants, animals or microorganisms. Enzymes originally isolated from microbes offer diverse advantages including ease of isolation, high consistency, higher yields, economic feasibility, high stability and candid production via recombinant DNA technology using microbes as host cells. Also the processes of product modification as well as optimization are easier for microbial enzymes as compared to those obtained from animals and plants. Thus microbial enzymes display enthralling features and opportunities, and form an important subclass of modern biopharmaceuticals. In this review, we focus on the curative potential of microbial enzymes including enzybiotics, digestive aids, anti-inflammatory, anti-cancer and fibrinolytic agents. This information will help to highlight and further explore their therapeutic potential which is fast gaining popularity and assisting healthcare.

Identification, characterization, and cloning of a novel aminoacylase, L-pipecolic acid acylase from Pseudomonas species.

L-Pipecolic acid is utilized as a vital component of specific chemical compounds, such as immunosuppressive drugs, anticancer reagents, and anesthetic reagents. We isolated and characterized a novel L-aminoacylase, N-acetyl-L-pipecolic acid-specific aminoacylase (LpipACY), from Pseudomonas sp. AK2. The subunit molecular mass of LpipACY was 45 kDa and was assumed to be a homooctamer in solution. The enzyme exhibited high substrate specificity toward N-acetyl-L-pipecolic acid and a high activity for N-acetyl-L-pipecolic acid and N-acetyl-L-proline. This enzyme was stable at a high temperature (60°C for 10 min) and under an alkaline pH (6.0-11.5). The N-terminal and internal amino acid sequences of the purified enzyme were STTANTLILRNG and IMASGGV, respectively. These sequences are highly consistent with those of uncharacterized proteins from Pseudomonas species, such as amidohydrolase and peptidase. We also cloned and overexpressed the gene coding LpipACY in Escherichia coli. Moreover, the recombinant LpipACY exhibited properties similar to native enzyme. Our results suggest that LpipACY is a potential enzyme for the enzymatic synthesis of L-pipecolic acid. This study provides the first description of the enzymatic characterization of L-pipecolic acid specific amino acid acylase.

Structures of l-asparaginase from Bacillus licheniformis Reveal an Essential Residue for its Substrate Stereoselectivity.

l-Asparaginase, which catalyzes the hydrolysis of l-asparagine, is an important enzyme in both the clinical and food industry. Exploration of efficient l-asparaginase with high substrate specificity, especially high chiral selectivity, is essential for extending its use. Herein, various crystal structures of type I l-asparaginase from Bacillus licheniformis (BlAsnase) have been resolved, and we found that there are two additional tyrosines in BlAsnase, contributing to the binding and catalysis of d-asparagine. Strikingly, the substitution of Tyr278 with methionine impaired the interaction with d-asparagine via water molecules due to the small hydrophobic side chain of methionine, which forced the ligand to the deep side of the active site toward the catalytic residues and thus resulted in the loss of hydrolyzing function. Our investigation of the substrate recognition mechanism of BlAsnase is significant for both a better understanding of l-asparaginase and its rational design to achieve high specificity for clinical and industrial applications.

Thermo stable tyrosinase purified from Pleurotus djamor grown in biomimetic calcium carbonate: A biological strategy to industrial waste remediation

A novel, neutral and heat-tolerant tyrosinase produced from Pleurotus djamor (P. djamor) using biomimetic calcium carbonate which was further used for the biodegradation of the phenolic waste of industrial effluents. Carbon-dioxide was sequestered/captured into calcium carbonate (CaCO 3 ) using carbonic anhydrase (2.1 U/mg protein) of Oceanobacillus with sequestration capacity as 24.7 CaCO 3 /mg protein in 25 s. This CaCO 3 was used as a substrate for cultivation of P. djamor (a mushroom) which was the potential source of tyrosinase. Mushroom tyrosinase was purified up to 51.7 folds purity which shown a molecular weight ∼ 90 kDa on SDS-PAGE. The optimal thermal and pH activity of the tyrosinase was observed at 50 °C and 7.0, respectively. The tyrosinase was stable within a pH range of 6.0–8.0, temperature 35 °C–55 °C and showed high substrate specificity for L-DOPA. Tyrosinase activity was drastically enhanced by Cu 2 + , Ca 2 + and Mn 2 + metal ions. K + ions were also found to inhibit the tyrosinase activity up to the moderate level. Mushroom tyrosinase was used to degrade phenol from industry effluents with 90% removal in 6 h. Extracellular nature as well as tyrosinase stability in the extreme conditions (temperature, pH, and presence of heavy metals) makes it preferable candidate for phenol bioremediation from industrial effluents. • Sequestration of carbon di oxide into calcium carbonate is clearly shown. • Biomimetically precipitated calcium carbonate is first time used for mushroom production. • Tyrosinase is purified from pre-mature phase of Pleurotus djamor . • Feasibility of degradation of phenol from industrial effluents by mushroom tyrosinase is presented. • A comprehensive mechanism for complete carbon-di-oxide utilization have been hypothesized.

The effect of heavy metals on thiocyanate biodegradation by an autotrophic microbial consortium enriched from mine tailings

Bioremediation systems represent an environmentally sustainable approach to degrading industrially generated thiocyanate (SCN−), with low energy demand and operational costs and high efficiency and substrate specificity. However, heavy metals present in mine tailings effluent may hamper process efficiency by poisoning thiocyanate-degrading microbial consortia. Here, we experimentally tested the tolerance of an autotrophic SCN−-degrading bacterial consortium enriched from gold mine tailings for Zn, Cu, Ni, Cr, and As. All of the selected metals inhibited SCN− biodegradation to different extents, depending on concentration. At pH of 7.8 and 30 °C, complete inhibition of SCN− biodegradation by Zn, Cu, Ni, and Cr occurred at 20, 5, 10, and 6 mg L−1, respectively. Lower concentrations of these metals decreased the rate of SCN− biodegradation, with relatively long lag times. Interestingly, the microbial consortium tolerated As even at 500 mg L−1, although both the rate and extent of SCN− biodegradation were affected. Potentially, the observed As tolerance could be explained by the origin of our microbial consortium in tailings derived from As-enriched gold ore (arsenopyrite). This study highlights the importance of considering metal co-contamination in bioreactor design and operation for SCN− bioremediation at mine sites.Key points• Both the efficiency and rate of SCN−biodegradation were inhibited by heavy metals, to different degrees depending on type and concentration of metal.• The autotrophic microbial consortium was capable of tolerating high concentrations of As, potential having adapted to higher As levels derived from the tailings source.

Biophysical analysis of the structural evolution of substrate specificity in RuBisCO

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant enzyme on Earth. However, its catalytic rate per molecule of protein is extremely slow and the binding of the primary substrate, CO2, is competitively displaced by O2. Hence, carbon fixation by RuBisCO is highly inefficient; indeed, in higher C3 plants, about 30% of the time the enzyme mistakes CO2 for O2 Using genomic and structural analysis, we identify regions around the catalytic site that play key roles in discriminating between CO2 and O2 Our analysis identified positively charged cavities directly around the active site, which are expanded as the enzyme evolved with higher substrate specificity. The residues that extend these cavities have recently been under selective pressure, indicating that larger charged pockets are a feature of modern RuBisCOs, enabling greater specificity for CO2 This paper identifies a key structural feature that enabled the enzyme to evolve improved CO2 sequestration in an oxygen-rich atmosphere and may guide the engineering of more efficient RuBisCOs.

Functional Implications of Second-Shell Basic Residues for dUTPase DR2231 Enzymatic Specificity

Nucleotide-processing enzymes are key players in biological processes. They often operate through high substrate specificity for catalysis. How such specificity is achieved is unclear. Here, we dea...

Specific hydrolysis of curdlan with a novel glycoside hydrolase family 128 β-1,3-endoglucanase containing a carbohydrate-binding module

The cbm6e gene from Saccharophagus degradans 2-40 T was cloned and expressed in Escherichia coli. CBM6E contains a glycoside hydrolase family 128 (GH128) catalytic module and a C-terminal carbohydrate-binding module (CBM) grouped into CBM family 6. The purified recombinant CBM6E displayed high substrate specificity toward curdlan as an endo-β-1,3-glucanase and had maximal activity at pH 6.0 and 35 ℃. The hydrolytic products against curdlan were predominantly laminaritriose (L3) and laminaritetraose (L4) along with a lower amount of laminaripentaose (L5) and laminarihexaose (L6). The CBM6 module selectively enhanced the enzyme activity against curdlan and displayed strict binding specificity to curdlan, no matter in its powder or high-set gel forms. This study laid a foundation for enzymatic degradation of curdlan to produce high-value β-1,3-glucooligosaccharides at moderate temperatures and provided a novel CBM tag for enzyme immobilization on curdlan.

High fungal substrate specificity limits the utility of environmental DNA to detect fungal diversity in bogs

Fungi are a highly diverse group of organisms and play a significant role in decomposition and carbon cycling in boreal ecosystems. To determine how fungal communities are structured in peat bogs and how to obtain representative samples for monitoring fungal community changes, we separately sampled and sequenced (ITS2, Illumina MiSeq) peat, mixed litter and litter from individual dominant plant species in five permanent plots in raised bogs in the Jizerské Hory Mountains (Czech Republic). In total, we detected 68–103 OTUs per plot. The fungal assemblages were mostly influenced by substrate identity, whereas the effect of the site was minimal. Only a few identified OTUs behave like generalists. The most specific fungi were found in dead parts of Calluna vulgaris, Trichophorum cespitosum and Drosera rotundifolia. The high substrate heterogeneity means that sequencing soil core samples does not provide an exhaustive inventory of the fungal diversity in bogs. Poor correspondence was observed between the recorded OTUs and either visually inspected fruitbodies or literature reports on the fungal taxa associated with the analysed substrates.