SUMMARY The secondary metabolome of Basidiomycota represents a largely uncharacterized source of pharmaceutically relevant natural products. Terpenoids are the primary class of bioactive compounds isolated from mushrooms. The Jack O’Lantern mushroom Omphalotus olearius was identified 50 years ago as a prolific producer of anticancer illudin sesquiterpenoids, however to date there have been exceptionally few studies into the biosynthesis of these important compounds. Here we report the draft genome sequence of O. olearius, which reveals a diverse network of sesquiterpene synthases and two metabolic gene clusters associated with illudin biosynthesis. Their biochemical characterization enabled a comprehensive survey of all currently available Basidiomycota genomes, thereby creating a predictive resource for terpenoid natural product biosynthesis in these organisms. Our results will facilitate discovery and biosynthetic production of unique pharmaceutically relevant bioactive compounds from Basidiomycota.

Basidiomycota represent a diverse source of natural products, particularly the sesquiterpenoids. Recently, the genome sequencing, mining, and subsequent discovery of a suite of sesquiterpene synthases was described in Omphalotus olearius. A predictive framework was developed to facilitate the discovery of sesquiterpene synthases in Basidiomycota. Phylogenetic analyses indicated a conservation of both sequence and initial cyclization mechanisms used. Here, the first robust application of this predictive framework is reported. It is used to pursue and selectively identify sesquiterpene synthases that follow a 1,6-, 1,10-, and 1,11-cyclization mechanism in the crust fungus Stereum hirsutum. The successful identification and characterization of a 1,6- and a 1,10-cyclizing sesquiterpene synthase, as well as three 1,11-cyclizing Δ-6 protoilludene synthases, is described. This study verifies the accuracy and utility of the predictive framework as a roadmap for the discovery of specific sesquiterpene synthases from Basidiomycota, representing an important step forward in natural product discovery.

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Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-␣1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-␣1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-␣1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-␣1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-␣1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases.Sesquiterpene synthases catalyze the cyclization of farnesyl pyrophosphate (FPP) to structurally diverse C 15 -hydrocarbons. These enzymes belong to the large group of terpene synthases that convert isoprene pyrophosphate substrates into hundreds of described terpenoid compounds by employing some of the most complex carbon-carbon forming reactions known (8). Many terpenoids are biologically active and are produced by plants, bacteria, and fungi such as, for example, antibiotics, toxins, and pheromones (5, 9).Catalysis in this class of enzymes is dependent on the presence of three Mg 2ϩ ions coordinated by two conserved motifs, an aspartate-rich DDXXD/E and an NSE/DTE motif, flanking the entrance of the active site. This Mg 2ϩ cluster binds the pyrophosphate (PP i ) group of FPP and positions the isoprenyl chain in the hydrophobic substrate binding pocket of the enzyme (10). Substrate binding triggers a conformational change that results in the closure of the active site and concurrent PP i cleavage to generate an initial transoid, allylic carbocation (6,7,35). This carbocation is then transferred along the isoprenyl chain and eventually quenched either by a water molecule or through proton abstraction. The binding pocket determines folding of the isoprenyl chain and chaperones the reactive carbocation intermediates until the final quenching step (20), thereby defining the product profile of a particular sesquiterpene synthase.Crystal structures have been solved for several microbial and plant sesquiterpene synthases (1,7,14,30). All enzymes share the same ␣-helical fold characteristic for ionization-dependent terpene synthases. Plant enzymes possess an additional, catalytically inactive N-terminal domain that h...

In this study we analyzed the structure and function of a truncated form of hemolysin A (HpmA265) from Proteus mirabilis using a series of functional and structural studies. Hemolysin A belongs to the two-partner secretion pathway. The two-partner secretion pathway has been identified as the most common protein secretion pathway among Gram-negative bacteria. Currently, the mechanism of action for the two-partner hemolysin members is not fully understood. In this study, hemolysis experiments revealed a unidirectional, cooperative, biphasic activity profile after full-length, inactive hemolysin A was seeded with truncated hemolysin A. We also solved the first x-ray structure of a TpsA hemolysin. The truncated hemolysin A formed a right-handed parallel ␤-helix with three adjoining segments of anti-parallel ␤-sheet. A CXXC disulfide bond, four buried solvent molecules, and a carboxyamide ladder were all located at the third complete ␤-helix coil. Replacement of the CXXC motif led to decreased activity and stability according to hemolysis and CD studies. Furthermore, the crystal structure revealed a sterically compatible, dry dimeric interface formed via anti-parallel ␤-sheet interactions between neighboring ␤-helix monomers. Laser scanning confocal microscopy further supported the unidirectional interconversion of full-length hemolysin A. From these results, a model has been proposed, where cooperative, ␤-strand interactions between HpmA265 and neighboring full-length hemolysin A molecules, facilitated in part by the highly conserved CXXC pattern, account for the template-assisted hemolysis.Hemolysin A (HpmA) 2 and B (HpmB) from Proteus mirabilis belong to the Type V b or two-partner secretion pathway (1), the most widespread of the five porin-type protein translocating systems found within bacterial, fungal, plant, and animal kingdoms (2). Cell surface adhesions, iron-acquisition proteins, and cytolysins/hemolysins all use two-partner secretion pathways (3-5). The A-component of the two-partner secretion in P. mirabilis is a 166-kDa virulence factor capable of mammalian blood cell lysis upon secretion from the cell. This is accomplished by Sec-dependent transport to the periplasm followed by N-terminal proteolytic processing. Extracellular secretion occurs by transport through the B-component, HpmB, which is a 16-stranded ␤-barrel transmembrane channel (6). In addition to its role in efficient secretion, HpmB is also necessary for activation of the larger exoprotein A-component (HpmA) (7-10).Studies on hemolytic TpsA members report that: 1) a truncated TpsA containing the N-terminal secretion cap (11) complements and restores hemolytic activity within a non-secreted/inactive pool of full-length TpsA (12), 2) the conserved cysteine residues within a CXXC motif are not required for secretion (12), and 3) the first asparagine within a NPNG hemagglutinin motif is required for efficient secretion (13). Other investigations demonstrate significant conformational change within TpsA members during B-component dependent sec...