Purification, crystallization and preliminary X-ray diffraction analysis of Omp6, a protoilludene synthase fromOmphalotus olearius

Abstract

Basidiomycetes produce a wide range of industrially relevant natural products. One of the main classes of natural products isolated from fungi are terpenoids, a highly diverse group of secondary metabolites, many of which are bioactive and have been adapted for pharmaceutical purposes. The discovery of a suite of novel sesquiterpene synthases from Omphalotus olearius via genome sequencing and bioinformatic analyses has recently been described. Here, the expression, purification and crystallization of one of these enzymes (Omp6), a protoilludene synthase, is reported. A native crystal diffracted to a resolution of 2.9 Å and belonged to space group P2 1 , with unit-cell parameters a = 43.67, b = 76.76, c = 107.22 Å , = = 90, = 95. A diffraction data set was collected on a home-source Rigaku/MSC MicroMax-007 X-ray generator.

Genome of Diaporthe sp. provides insights into the potential inter-phylum transfer of a fungal sesquiterpenoid biosynthetic pathway

“…To identify genes encoding putative sesquiterpene synthases in BR109 we carried out BLAST searches using the sequences of diverse fungal sesquiterpene synthases previously cloned and characterized by our group: Cop1–6 from the Basidiomycete Coprinus cinereus (Agger et al, 2009; Lopez-Gallego et al, 2010a; Lopez-Gallego et al, 2010b); Omp1–11 from the Basidiomycete Omphalotus olearius (Quin et al, 2013b; Wawrzyn et al, 2012b); and Stehi159379, Stehi128017, Stehi25180, Stehi64702 and Stehi73029 from the Basidiomycete Stereum hirsutum (Quin et al, 2013a; Quin et al, 2015). We also included sequences of sesquiterpene synthases characterized from Ascomycetes by other groups: presilphiperfolan-8β-ol synthase BcBOT2 from Botrytis cinerea (Wang et al, 2009), koraiol synthase Ffsc4 and acorenol synthase Ffsc6 from Fusarium fujikori (Brock et al, 2013), trichodiene synthase FsTS from Fusarium sporotrichioides (Hohn and Vanmiddlesworth, 1986), longiborneol synthase FgCLM1 from Fusarium graminearium (McCormick et al, 2010), aristolochene synthase PrAS from Penicillium roqueforti (Caruthers et al, 2000), as well as 11 terpene synthases Hyp1–11 recently identified in Hypoxylon sp.…”

Traversing the fungal terpenome

“…Omp6 and Omp7 cyclize FPP highly selective into Δ6-protoilludene 29 (Scheme 4) while the other enzymes also display new cyclization activities, producing barbatenes 12, 35 (Omp9) and daucenes 36, 37 (Omp 10). 23, 76 Another Δ6-protoilludene synthase was cloned from the honey mushroom Armillaria gallica (ArmGa1) which makes antimicrobial melleolide I 38 (Scheme 6) sesquiterpenoids. 77 …”

“…The biochemical characterization of the identified sesquiterpene synthases from Coprinus cinereus (Cop1–6) 69–71 and of Omphalotus olearius (Omp1–10) 23, 76 guided the subsequent development of in silico approaches for the directed discovery of new sesquiterpene synthases and their associated biosynthetic genes based upon cyclization mechanism of choice. 20 Initiatives such as the 1000 Fungal Genomes Project 78 have led to a substantial increase in the number of sequenced genomes that are publically available.…”

Moonlighting Metals: Insights into Regulation of Cyclization Pathways in Fungal Δ6‐Protoilludene Sesquiterpene Synthases

“…These enzymes perform a 1,11 cyclization mechanism to produce the trans ‐humulyl cation ( 7 )‐derived Δ 6 ‐protoilludene ( 1 ; Scheme ). When characterized under typical reaction conditions, the purified enzymes were highly active and produced exclusively 1 (Scheme ) . The exquisite product fidelity of these enzymes contrasts with the majority of characterized sesquiterpene synthases, which typically synthesize several products by the same cyclization pathway.…”

“…Surprisingly, when expressed heterologously, Stehi64702 and Stehi73029 were capable of following dual cyclization mechanisms, thereby resulting in mainly the 1,11 cyclization product 1 but also low levels of the 1,10 cyclization product germacrene A, detected as the heat‐induced Cope rearrangement product β‐elemene ( 2 ; Scheme ) . Previous characterization of Omp6 and Omp7 showed that they were highly specific and that their product specificity was not altered by site‐directed mutagenesis of active‐site residues, nor by varying pH, temperature or concentration of NaCl, Mg 2+ , and Mn 2+ ions . It was therefore unexpected that the closely related homologues Stehi64702 and Stehi73029 were less specific when expressed heterologously .…”