An Enzyme Catalyzing O‐Prenylation of the Glucose Moiety of Fusicoccin A, a Diterpene Glucoside Produced by the Fungus Phomopsis amygdali

Abstract

Isoprenoids form the largest family of compounds found in nature. Isoprenoids are often attached to other moieties such as aromatic compounds, indoles/tryptophan, and flavonoids. These reactions are catalyzed by three phylogenetically distinct prenyltransferases: soluble aromatic prenyltransferases identified mainly in actinobacteria, soluble indole prenyltransferases mostly in fungi, and membrane-bound prenyltransferases in various organisms. Fusicoccin A (FC A) is a diterpene glycoside produced by the plant-pathogenic fungus Phomopsis amygdali and has a unique O-prenylated glucose moiety. In this study, we identified for the first time, from a genome database of P. amygdali, a gene (papt) encoding a prenyltransferase that reversibly transfers dimethylallyl diphosphate (DMAPP) to the 6'-hydroxy group of the glucose moiety of FC A to yield an O-prenylated sugar. An in vitro assay with a recombinant enzyme was also developed. Detailed analyses with recombinant PAPT showed that the enzyme is likely to be a monomer and requires no divalent cations. The optimum pH and temperature were 8.0 and 50 °C, respectively. K(m) values were calculated as 0.49±0.037 μM for FC P (a plausible intermediate of FC A biosynthesis) and 8.3±0.63 μM for DMAPP, with a k(cat) of 55.3±3.3×10⁻³ s. The enzyme did not act on representative substrates of the above-mentioned three types of prenyltransferase, but showed a weak transfer activity of geranyl diphosphate to FC P.

Biosynthesis of Terpenoid Natural Products in Fungi

“…xanthones) [66,67] or to O-and N-functional groups of aromatic moieties [68,69]. More recently, an enzyme was identified that catalyzes O-prenylation of a glucose moiety of the diterpene fusicoccin A [70]. Mechanistic and structural studies revealed that these proteins adopt a new β-barrel fold containing repeating αββα-secondary structure elements, which gives this enzyme family their name: ABBA-type.…”

“…8). This cluster was identified in the draft genome sequence of P. amygdali based on a previously identified ABBA-type prenyltransferases that catalyzed in vitro the reverse O-prenylation of glucose with DMAPP [70], which is a prenylation that is also present in fusiccocin A. A combination of in vitro assays with recombinant enzymes and gene disruption allowed Dairi's group to then establish the complete fusicoccin A pathway [189].…”

A fungal prenyltransferase catalyzes the regular di-prenylation at positions 20 and 21 of paxilline

“…23) Mycelia of the transformants were inoculated into a medium (3.5% Czapek-Dox, 2% starch, 1% polypeptone; final volume 100 mL) and cultivated at 30°C for 3 d in a reciprocal shaker (125 strokes/min). cDNA preparation was performed by a method described previously, 30) with the primer set shown in Supplemental Table 1.…”

Genome Mining for Fungal Secondary Metabolic Gene Clusters

“…These include the well-studied gibberellin pathways found in several fungi that use a bifunctional diterpene synthase that combines the domains of a class I and class II terpene synthases (which are separate enzymes in plant diterpene biosynthesis) to cyclize GGPP [111][112][113]. Mining of Ascomycota genomes followed by gene deletion studies and stepwise heterologous coexpression of pathway genes in Aspergillus has led to the elucidation of additional gene clusters involved in the biosynthesis of the diterpene compounds fusicoccin, brassicicene, aphidicolin and phomopsene [54,55,[114][115][116][117][118][119][120], and a sesterterppenoid (C 25 ) [121]. Intriguingly, these terpenoid scaffolds are built by a novel-type of chimeric terpene synthases that combines the domains of a class I terpene synthase and a prenyldiphosphate synthase that provides the C 25 isoprene substrate for the cyclase [55].…”