A Nonheme Fe^II/2-Oxoglutarate-Dependent Oxygenase Catalyzes a Double Bond Migration within a Dimethylallyl Moiety Accompanied by Hydroxylation

Abstract

Prenylation of cyclodipeptides contributes largely to the structure diversification and biological activity. The prenylated products can be further metabolized by modifications like hydroxylation with cytochrome P450 enzymes or nonheme Fe/2-oxoglutarate-dependent oxygenases. Herein, we cloned and overexpressed NFIA_045530 from Neosartorya fischeri, which shares high sequence similarity with the nonheme Fe/2-oxoglutarate-dependent oxygenase FtmOx1 from Aspergillus fumigatus on the amino acid level. FtmOx1 is a member of the biosynthetic enzymes for fumitremorgin-type mycotoxins and catalyzes the conversion of fumitremorgin B to verruculogen by insertion of an oxygen molecule into the two prenyl moieties. The recombinant protein EAW25734 encoded by NFIA_045530 was purified to apparent homogeneity and then was used for incubation with intermediates of the fumitremorgin biosynthetic pathway. LC-MS analysis revealed no consumption of fumitremorgin B but good conversion with its biosynthetic precursor tryprostatin B in the presence of Fe and 2-oxoglutarate. Structure elucidation confirmed 22-hydroxylisotryprostatin B and 14α, 22-dihydroxylisotryprostatin B as the major enzyme products. Further detailed biochemical characterization led to the identification of a novel enzyme, which catalyzes a double bond migration within the dimethylallyl moiety of tryprostatin B with concomitant hydroxylation. Incubation with O-enriched atmosphere confirmed O as the major origin of the hydroxyl groups. Solvent exchange was also observed for that at C22. LC-MS analysis confirmed the presence of 22-hydroxylisotryprostatin B in a Neosartorya fischeri extract, highlighting the role of this enzyme in the metabolism of intermediates of the fumitremorgin/verruculogen pathway. A plausible reaction mechanism implementing a radical rearrangement prior to accepting a hydroxyl radical from Fe is discussed.

Properties of the Reactants and Their Interactions within and with the Enzyme Binding Cavity Determine Reaction Selectivities. The Case of Fe(II)/2‐Oxoglutarate Dependent Enzymes

“…Their biological roles span from regulation of gene expression, [4,5] repair and modification of nucleic acids, [6,7] fatty acid metabolism, [8] and collagen biosynthesis to biosynthesis of secondary metabolites, [9] among them pharmacologically active compounds [10] . The latter are formed by ODDs from plants and microorganisms and contain double bonds, halogen substituents or rings [11–17] . The versatility of ODDs raises quite a lot of interest, therefore the scope of the catalysed reactions, [11,18–21] the structural and mechanistic insights into ODDs activity, [22–26] as well as their industrial applications were widely discussed [10,27] …”

“…[10] The latter are formed by ODDs from plants and microorganisms and contain double bonds, halogen substituents or rings. [11][12][13][14][15][16][17] The versatility of ODDs raises quite a lot of interest, therefore the scope of the catalysed reactions, [11,[18][19][20][21] the structural and mechanistic insights into ODDs activity, [22][23][24][25][26] as well as their industrial applications were widely discussed. [10,27] The overall structure of ODDs is based on a double stranded beta helix (DSBH) fold forming a jelly roll barrel (see Figure 1A).…”

Genomic Locus of a Penicillium crustosum Pigment as an Integration Site for Secondary Metabolite Gene Expression

“…For LC–MS analysis, 5 μL samples were injected into an Agilent 1260 series HPLC system (Agilent) combined with a Bruker microTOF QIII mass spectrometer under conditions described previously …”

“…For LC−MS analysis, 5 μL samples were injected into an Agilent 1260 series HPLC system (Agilent) combined with a Bruker microTOF QIII mass spectrometer under conditions described previously. 43 1 H NMR spectra were taken on a JEOL ECA-500 MHz spectrometer (JEOL) and processed with the software MestReNova 6.1.0 (Metrelab). Signals of the solvents were used as reference of the chemical shifts.…”

Peniphenone and Penilactone Formation in Penicillium crustosum via 1,4-Michael Additions of ortho-Quinone Methide from Hydroxyclavatol to γ-Butyrolactones from Crustosic Acid

“…For biochemical characterization, claD was amplified and cloned into pET28a (+). The purified ClaD (Figure S23) was used for incubation with 5 in the presence of ascorbate (AA), Fe­[(NH 4 ) 2 (SO 4 ) 2 ], and 2-oxoglutarate (2OG). , HPLC analysis confirmed the oxidation of 5 to 9 with a conversion yield of 22.5% after incubation with 2 μg protein at 37 °C for 30 min (Figure A). Nearly no consumption of 5 was detected in the assays without ascorbate or 2-oxoglutarate.…”