A Novel Tyrosinase from Armillaria ostoyae with Comparable Monophenolase and Diphenolase Activities Suffers Substrate Inhibition.

Tyrosinase is a bifunctional enzyme mediating the o-hydroxylation and two-electron oxidation of monophenols to o-quinones. The monophenolase activity of tyrosinase is much desired for the industrial synthesis of catechols. However, the generally low ratio of monophenolase/diphenolase activity of tyrosinase limited its utilization in the industry. In this study, a novel tyrosinase from Armillaria ostoyae strain C18/9 (AoTyr) was characterized, and the results showed that the enzyme has an optimal temperature of 25°C and an optimal pH of 6. The enzyme has comparable monophenolase and diphenolase activities and exhibits substrate inhibition in both of the activities. In silico analysis and mutagenesis experiments showed that residues 262 and 266 play important roles in modulating the substrate inhibition and enzymatic activities of AoTyr, and the replacement of D262 with asparagine significantly increased the monophenolase/diphenolase catalytic efficiencies (kcat/Km ratios) (1.63-fold) of the enzyme. The results from this study indicated that this novel tyrosinase could be a potential candidate for the industrial biosynthesis of catechols. IMPORTANCE Tyrosinase is able to oxidize various phenolic compounds, and its ability to convert monophenols into diphenols has caught great attention in the research field and industrial applications. However, the utilization of tyrosinase for the industrial synthesis of catechols has been limited due to the fact that the monophenolase activity of most of the known tyrosinases is much lower than the diphenolase activity. In the present study, a novel tyrosinase with comparable monophenolase and diphenolase activities was characterized. The enzyme exhibits substrate inhibition in both monophenolase and diphenolase activities. In silico analysis followed by mutagenesis experiments confirmed the important roles of residues 262 and 266 in the substrate inhibition and activity modulation of the enzyme, and the D262N variant showed an enhanced monophenolase/diphenolase catalytic efficiency ratio compared to the wild-type enzyme.

Oxidase or peptidase? A computational insight into a putative aflatoxin oxidase from Armillariella tabescens.

Aflatoxin oxidase (AFO), an enzyme isolated from Armillariella tabescens, has been reported to degrade aflatoxin B1 (AFB1). However, recent studies reported sequence and structure similarities with the dipeptidyl peptidase III (DPP III) family of enzymes and confirmed peptidase activity toward DPP III substrates. In light of these investigations, an extensive computational study was performed in order to improve understanding of the AFO functions. Steered MD simulations revealed long-range domain motions described as protein opening, characteristic for DPPs III and necessary for substrate binding. Newly identified open and partially open forms of the enzyme closely resemble those of the human DPP III orthologue. Docking of a synthetic DPP III substrate Arg2 -2-naphthylamide revealed a binding mode similar to the one found in crystal structures of human DPP III complexes with peptides with the S1 and S2 subsites' amino acid residues conserved. On the other hand, no energetically favorable AFB1 binding mode was detected, suggesting that aflatoxins are not good substrates of AFO. High plasticity of the zinc ion coordination sphere within the active site, consistent with that of up to date studied DPPs III, was observed as well. A detailed electrostatic analysis of the active site revealed a predominance of negatively charged regions, unsuitable for the binding of the neutral AFB1. The present study is in line with the most recent experimental study on this enzyme, both suggesting that AFO is a typical member of the DPP III family.

Novel Application of Peptidyl-Lys Metallopeptidase as a C-Terminal Processing Protease.

Adding fusion partners to proteins or peptides can aid or be a necessity to facilitate recombinant expression, folding, or purification. Independent of the reason it is desirable to remove the fusion partner to restore native functionality. Processing proteases catalyze the removal of fusion partners, however, most of these proteases have substrate specificity for the N-terminal of the scissile bond, leaving non-native termini if fusions are added to the C-terminal. The peptidyl-lys metallopeptidease of Armillaria mellea (Am-LysN) is unusual by having substrate specificity for the C-terminal side of the scissile peptide bond, allowing it to generate native C-termini. Am-LysN has strict specificity for lysine in P1', making all lysines of a protein or peptide a potential degradation site, however there are a number of amino acid side chains which lower hydrolysis significantly when located adjacent to the lysine. In this study we show that Am-LysN can be used as a processing protease to remove C-terminal extensions of peptides with no internal lysine to generate native Ctermini. Furthermore we show that removal of C-terminal extensions on peptides containing internal lysines can be achieved with little degradation of the product depending on the adjacent amino acids. These results demonstrate the utility of LysN allowing for novel ways to use fusion technology in the production of recombinant proteins.

Heterologous expression of peptidyl-Lys metallopeptidase of Armillaria mellea and mutagenic analysis of the recombinant peptidase.

A method to express, purify and modify the Peptidyl-Lys metallopeptidase (LysN) ofArmillaria melleainPichia pastoriswas developed to enable functional studies of the protease. Based on prior work, we propose a mechanism of action of LysN. Catalytic residues were investigated by site-directed mutagenesis. As anticipated, these mutations resulted in significantly reduced catalytic rates. Additionally, based on molecular modelling eleven mutants were designed to have altered substrate specificity. The S1' binding pocket of LysN is quite narrow and lined with negative charge to specifically accommodate lysine. To allow for arginine specificity in S1', it was proposed to extend the S1' binding pocket by mutagenesis, however the resulting mutant did not show any activity with arginine in P1'. Two mutants, A101D and T105D, showed increased specificity towards arginine in subsites S2'-S4' compared to the wild type protease. We speculate that the increased specificity to result from the additional negative charge which attract and interact with positively charged residues better than the wild type.

A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins.

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1 to ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A 5-fold, secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier-protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share an ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.

Saccharification of sunflower stalks using lignocellulases from a fungal consortium comprising Pholiota adiposa and Armillaria gemina.

Lignocellulases from Armillaria gemina and Pholiota adiposa are efficient in hydrolyzing aspen and poplar biomass, respectively. In the present study, lignocellulosic enzymes obtained from a fungal consortium comprising P. adiposa and A. gemina were used for the saccharification of sunflower stalks. Sunflower stalks were thermochemically pretreated using 2 % NaOH at 50 °C for 24 h. The saccharification process parameters including substrate concentration, enzyme loading, pH, and temperature were optimized using response surface methodology to improve the saccharification yield. The highest enzymatic hydrolysis (84.3 %) was obtained using the following conditions: enzyme loading 10 FPU/g-substrate, substrate 5.5 %, temperature 50 °C, and pH 4.5. The hydrolysis yield obtained using the enzymes from the fungal consortium was equivalent to that obtained using a mixture of commercial enzymes Celluclast and Novozyme ß-glucosidase. Addition of up to 500 ppm of heavy metal ions (As, Cu, Fe, Mn, Ni, Pb, and Zn) during saccharification did not significantly affect the saccharification yield. Thus, the biomass grown for phytoremediation of heavy metals can be used for the production of reducing sugars followed by ethanol fermentation.

Substrate Specificity Profiling of Peptidyl-Lys Metallopeptidase of Armillaria mellea by FRET Based Peptide Library.

Determining the substrate specificity of a protease is essential for developing assays, inhibitors and understanding the mechanisms of the enzyme. In this work, we have profiled the specificity of Peptidyl-Lys metallopeptidase, (LysN), of Armillaria mellea, by a synthetic fluorescence resonance energy transfer (FRET) positional-scanning library. The library was based on a reference sequence K(Abz)-S-A-Q-K-M-V-S-K(Dnp), where the fluorescent donor is 2-aminobenzamide and the quencher is N-2,4-dinitrophenyl. Each position was varied between 19 different amino acids one by one, to reveal the specificity of the protease. LysN exhibits strict specificity for lysine in S1', and has less specificity moving further away from the scissile bond. Additivity between the subsites was observed and the best substrate identified was K(Abz)-M-R-F-K-R-R-R-K(Dnp) with a kcat/KM of 42.6 µM/s. Based on a homology structure model the reference substrate was fitted into the active site using molecular dynamics to propose peptide-enzyme interactions.

The furofuran-ring selectivity, hydrogen peroxide-production and low Km value are the three elements for highly effective detoxification of aflatoxin oxidase.

AFO (aflatoxin oxidase), an enzyme from Armillariella tabescens previously named aflatoxin detoxifizyme, exhibits oxidative detoxification activity toward aflatoxin B1 and sterigmatocystin. Bioinformatics reveals that AFO is a newly discovered oxidase because AFO does not share any significant similarities with any known oxidase. It is critically important to understand how AFO acts on aflatoxin B1. In this study, in addition to aflatoxin B1 (AFB1) and sterigmatocystin (ST), five other chemicals that have furan or pyran structures were investigated. The results indicated that in addition to AFB1 and ST, AFO is also able to act on versicolorin A, 3,4-dihydro-2H-pyran and furan. These results suggested that 8,9-unsaturated carboncarbon bond of aflatoxin B1 is the potential reactive site for AFO. Further findings indicated that the action of AFO is oxygen-dependent and hydrogen peroxide-producing. The simultaneously produced-hydrogen peroxide possibly plays the essential role in detoxification of AFO. In addition, the extremely low Km value of 0.33 µmol/l for AFO-AFB1 and 0.11 µmol/l for AFO-ST signifies that AFO is highly selective for AFB1 as well as ST.

Characterization of a novel endo-ß-1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis.

A novel endo-ß-1,4-glucanase (EG)-producing strain was isolated and identified as Armillaria gemina KJS114 based on its morphology and internal transcribed spacer rDNA gene sequence. A. gemina EG (AgEG) was purified using a single-step purification by gel filtration. The relative molecular mass of AgEG by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 65 kDa and by size exclusion chromatography was 66 kDa, indicating that the enzyme is a monomer in solution. The pH and temperature optima for hydrolysis were 5.0 and 60 °C, respectively. Purified AgEG had the highest catalytic efficiency with carboxymethylcellulose (k(cat)/K(m) = 3,590 mg mL⁻¹ s⁻¹) unlike that reported for any fungal EG, highlighting the significance of the current study. The amino acid sequence of AgEG showed homology with hydrolases from the glycoside hydrolase family 61. The addition of AgEG to a Populus nigra hydrolysate reaction containing a commercial cellulase mixture (Celluclast 1.5L and Novozyme 188) showed a stimulatory effect on reducing sugar production. AgEG is a good candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.

Assembly of melleolide antibiotics involves a polyketide synthase with cross-coupling activity.

Little is known about polyketide biosynthesis in mushrooms (basidiomycota). In this study, we investigated the iterative type I polyketide synthase (PKS) ArmB of the tree pathogen Armillaria mellea, a producer of cytotoxic melleolides (i.e., polyketides esterified with various sesquiterpene alcohols). Heterologously produced ArmB showed orsellinic acid (OA) synthase activity in vitro. Further, we demonstrate cross-coupling activity of ArmB, which forms OA esters with various alcohols. Using a tricyclic Armillaria sesquiterpene alcohol, we reconstituted the biosynthesis of melledonol. Intermolecular transesterification reactions may represent a general mechanism of fungal PKSs to create structural diversity of small molecules. Phylogenetic network construction of thioesterase domains of both basidiomycetes and ascomycetes suggests that the fungal nonreducing PKS family has likely evolved from an ancient OA synthase and has gained versatility by adopting Claisen-like cyclase or transferase activity.

Enzymatic hydrolysis of aspen biomass into fermentable sugars by using lignocellulases from Armillaria gemina.

A white rot fungus, identified as Armillaria gemina SKU2114 on the basis of morphological and phylogenetic analyses, was found to secrete efficient lignocellulose-degrading enzymes. The strain showed maximum endoglucanase, cellobiohydrolase, and ß-glucosidase activities of 146, 34, and 15 U/mL, respectively, and also secreted xylanase, laccase, mannanase, and lignin peroxidase with activities of 1270, 0.16, 57, and 0.31 U/mL, respectively, when grown with rice straw as a carbon source. Among various plant biomasses tested for saccharification, aspen biomass produced the maximum amount of reducing sugar. Response surface methodology was used to optimize the hydrolysis of aspen biomass to achieve the highest level of sugar production. A maximum saccharification yield of 62% (429 mg/g-substrate) was obtained using Populus tomentiglandulosa biomass after 48 h of hydrolysis. A. gemina was shown to be a good option for use in the production of reducing sugars from lignocellulosic biomass.

Degradation and transformation of anthracene by white-rot fungus Armillaria sp. F022.

Characterization of anthracene metabolites produced by Armillaria sp. F022 was performed in the enzymatic system. The fungal culture was conducted in 100-mL Erlenmeyer flask containing mineral salt broth medium (20 mL) and incubated at 120 rpm for 5-30 days. The culture broth was then centrifuged at 10,000 rpm for 45 min to obtain the extract. Additionally, the effect of glucose consumption, laccase activity, and biomass production in degradation of anthracene were also investigated. Approximately, 92 % of the initial concentration of anthracene was degraded within 30 days of incubation. Dynamic pattern of the biomass production was affected the laccase activity during the experiment. The biomass of the fungus increased with the increasing of laccase activity. The isolation and characterization of four metabolites indicated that the structure of anthracene was transformed by Armillaria sp. F022 in two routes. First, anthracene was oxidized to form anthraquinone, benzoic acid, and second, converted into other products, 2-hydroxy-3-naphthoic acid and coumarin. Gas chromatography-mass spectrometry analysis also revealed that the molecular structure of anthracene was transformed by the action of the enzyme, generating a series of intermediate compounds such as anthraquinone by ring-cleavage reactions. The ligninolytic enzymes expecially free extracellular laccase played an important role in the transformation of anthracene during degradation period.

A rational design for trypsin-resistant improvement of Armillariella tabescens ß-mannanase MAN47 based on molecular structure evaluation.

Protease resistance of enzymes is required for the feed industry because of the presence of secretary proteases in the digestive tract. In this study, we report a rational method for protease-resistance improvement of enzymes based on molecular structure evaluation. The trypsin-resistance of ß-mannanase MAN47 from Armillariella tabescens was investigated. Twelve tryptic sites within it were ordered by their positions in three-dimensional space from external to internal. Except of R144, R192 and R261, which were either conserved or highly related to the catalytic activity, the top external residue K280 and the most internal residue K371 were selected. With conducting computational design via H-bond analysis and molecular dynamics simulations, optimal mutants of K280N and K371Q were predicted. Meanwhile, a triple mutant K280N/K107H/R102N was also predicted. Half-lives of mutants K280N, K280N/K107H/R102N, K371Q and wild-type enzymes which were all pre-treated by trypsin at 40 °C were determined 185 min, 285 min, 102 min and 100 min, respectively. In addition, the temperature-activity and pH-activity profiles revealed that the mutations we designed had no obvious influence on the catalytic activity of the enzyme. Our results proved that trypsin-resistance of an enzyme could be improved by molecular rational evolution of homology modeling and molecular dynamics simulations.

Characterization of a novel xylanase from Armillaria gemina and its immobilization onto SiO2 nanoparticles.

Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and ß-glucosidase activities of 1,270, 146, 34, and 15 U mL(-1), respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k (cat)/K (m) = 1,440 mg mL(-1) s(-1)) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117 % immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8 % higher production of xylooligosaccharides compared to free enzyme. After 17 cycles, the immobilized enzyme retained 92 % of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.

Identification of naphthalene metabolism by white rot fungus Armillaria sp. F022.

Armillaria sp. F022, a white rot fungus isolated from tropical rain forest (Samarinda, Indonesia) was used to biodegrade naphthalene in cultured medium. Transformation of naphthalene by Armillaria sp. F022 which is able to use naphthalene, a two ring-polycyclic aromatic hydrocarbon (PAH) as a source of carbon and energy was investigated. The metabolic pathway was elucidated by identifying metabolites, biotransformation studies and monitoring enzyme activities in cell-free extracts. The identification of metabolites suggests that Armillaria sp. F022 initiates its attack on naphthalene by dioxygenation at its C-1 and C-4 positions to give 1,4-naphthoquinone. The intermediate 2-hydroxybenzaldehyde and salicylic acid, and the characteristic of the meta-cleavage of the resulting diol were identified in the long-term incubation. A part from typical metabolites of naphthalene degradation known from mesophiles, benzoic acid was identified as the next intermediate for the naphthalene pathway of this Armillaria sp. F022. Neither phthalic acid, catechol and cis,cis-muconic acid metabolites were detected in culture extracts. Several enzymes (manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase and 2,3-dioxygenase) produced by Armillaria sp. F022 were detected during the incubation.

The novel role of fungal intracellular laccase: used to screen hybrids between Hypsizigus marmoreus and Clitocybe maxima by protoplasmic fusion.

Laccase has been proved important in decolorization of Remazol Brilliant Blue R (RBBR), oxidation of 2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, lignin degradation and fruiting-body formation. The decolorization of RBBR by laccase was firstly used to screen protoplast fusants. Fusants were obtained by protoplast fusion between the strains of Hypsizigus marmoreus and Clitocybe maxima, and two fusants (IM1 and IIIM5) were screened on PDA medium containing RBBR. These fusants were significant higher in laccase activity than H. marmoreus, nearly 413 and 395 times, respectively. Their hyphal growth rates were also remarkable higher than H. marmoreus, nearly 1.5 and 1.4 times, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed these fusants contained the laccase, and the molecular mass of the laccase was consistent with the laccase of C. maxima, nearly 62 kDa. The pileus color of the IM1 and IIIM5 also showed partial recombined characteristics comparing to the parental strains, while biological efficiency ratios were prominent higher than that of H. marmoreus, up to 14.58 and 10.87 %, respectively. Randomly amplified polymorphic DNA bands of fusants not only were similar to parental bands, but presented new non-parental bands. Using the Unweighted pair-group method together with mathematic averages method to gain a dendrogram, in which the fusants showed intra-cluster variations. Significantly, H. marmoreus was the dominant parent, while C. maxima were distant from the fusants. The differences among IM1, IIIM5 and H. marmoreus, and the similarities among IM1, IIIM5 and C. maxima indicated IM1 and IIIM5 were somatic hybrids of H. marmoreus and C. maxima. Accordingly, it is feasible to use laccase to screen fusants of H. marmoreus and C. maxima.

Cloning, expression, purification and characterization of an aflatoxin-converting enzyme from Armillaria tabescens.

OBJECTIVE: Aflatoxin B1 (AFB1) is extremely mutagenic, toxic and a potent carcinogen both to humans and livestock. Aflatoxin-oxidase (AFO) was an aflatoxin-converting enzyme previously purified by us from Armillaria tabescens. In order to know better about the molecular characterization of this distinct enzyme, we expressed, purified and characterized the His6 tag fused aflatoxin-oxidase. METHODS: Based on sequences of peptides fragments of AFO previously obtained by Electrophoresis-Electrospray Ionization tandem mass spectrometry (ESI-MS/MS), we cloned the cDNA of AFO using Switching Mechanism At 5' end of the RNA Transcript (SMART) Rapid Amplification of cDNA Ends (RACE) technology and expressed this gene as a fusion protein in Pichia pastoris by using pPIC9-afo as vector. We purified the fusion enzyme using nickel affinity chromatography. We identified the recombinant aflatoxin-oxidase (rAFO) by both western blot and peptide mass fingerprinting (PMF). Moreover, we characterized several enzymatic properties of the rAFO using AFB1 as the substrate including Km value, optimum temperature, optimum pH, thermal stability and pH stability. RESULTS: The AFO gene is 2321 bp long with a coding region of 2088 bp encoding 695 amino acids. Peptide mass fingerprinting (PMF) identification showed a 63.2% coverage of the molecule compared to the theoretical tryptic cleavage of the rAFO. The recombinant aflatoxin oxidase was purified 5.99-folds using nickel affinity chromatography. It has a specific activity of 234 U/mg. Kinetics studies showed that the rAFO converted AFB1 with the Km value of 3.93 +/- 0.20 x 10(-6) mol/L under its optimal conditions of pH 6.0 and 30 degrees C. Thermostability investigation revealed that the rAFO had a half-life of 90 min at 30 degrees C, and pH stability results suggested that the rAFO was relatively stable when pH ranged from 5.5 to 7.5. CONCLUSION: It appears to be the first successful production of the recombinant aflatoxin oxidase (rAFO) with AFB1-converting ability from Armillaria tabescens. The purified rAFO with preferably AFB1-converting activity confirms that this recombinant aflatoxin oxidase is now ready for further studying.

Characterisation of the ArmA adenylation domain implies a more diverse secondary metabolism in the genus Armillaria.

The armA-gene, encoding a tridomain enzyme reminiscent of nonribosomal peptide synthetases, was identified in the genome of the basidiomycete Armillaria mellea. Heterologously expressed enzyme and the ATP-pyrophosphate exchange assay were used for the in vitro biochemical characterisation of the ArmA adenylation domain. l-leucine was the preferred substrate, while l-threonine, l-valine, l-alanine, and l-isoleucine were turned over at lower rates (83 %, 62 %, 56 %, and 44 %, respectively). Other proteinogenic amino acids, 2-oxo acids, and benzoic acid derivatives were not accepted. As the substrate range of ArmA is incompatible with the secondary metabolites known from the genus Armillaria, our results imply greater natural product diversity in this genus. This is the first biochemical characterisation of a basidiomycete amino acid-adenylating domain, and our results may help refine computer algorithms to predict substrate specificities for basidiomycete nonribosomal peptide synthetases whose genes are discovered through genome sequencing.

Phospholipid acylhydrolases trigger membrane degradation during fungal sporogenesis.

Armillaria ostoyae is a phytopathogen infecting coniferous trees. Fruiting bodies of this basidiomycete contain high phospholipase A(1) (PLA(1)) activity. In this paper, the role of phospholipid-deacylating activity, which was also detected in fruiting bodies of other basidiomycetes, in the fungal lipid metabolism is elucidated. For A. ostoyae the occurrence of PLA(1) activity is shown to be restricted to the late reproductive phase, correlating with the release of mature spores. Specific expression in the spore-producing tissue provides evidence for the involvement of PLA(1) in spore formation. Based on lipid analysis, the degradation of membrane phospholipids in this tissue can be ascribed mainly to PLA(1) activity because other enzymes such as phospholipases C and D, triglyceride lipase and phosphatidic acid phosphatase had only low activities. A concomitant increase in the concentration of fatty acids and their anabolites (di- and triglycerides), which are used as storage lipids in the developing fungal spore cells, was observed. Therefore, PLA(1) contributes to the formation of spores by providing membrane constituents as a source of fatty acids.

Cloning and characterization of an Armillaria gallica cDNA encoding protoilludene synthase, which catalyzes the first committed step in the synthesis of antimicrobial melleolides.

Melleolides and related fungal sesquiterpenoid aryl esters are antimicrobial and cytotoxic natural products derived from cultures of the Homobasidiomycetes genus Armillaria. The initial step in the biosynthesis of all melleolides involves cyclization of the universal sesquiterpene precursor farnesyl diphosphate to produce protoilludene, a reaction catalyzed by protoilludene synthase. We achieved the partial purification of protoilludene synthase from a mycelial culture of Armillaria gallica and found that 6-protoilludene was its exclusive reaction product. Therefore, a further isomerization reaction is necessary to convert the 6-7 double bond into the 7-8 double bond found in melleolides. We expressed an A. gallica protoilludene synthase cDNA in Escherichia coli, and this also led to the exclusive production of 6-protoilludene. Sequence comparison of the isolated sesquiterpene synthase revealed a distant relationship to other fungal terpene synthases. The isolation of the genomic sequence identified the 6-protoilludene synthase to be present as a single copy gene in the genome of A. gallica, possessing an open reading frame interrupted with eight introns.