Hispidospermidin

The fungal species Aspergillus flavus produces an alkaloid terpenoid, flavunoidine, through a hybrid biosynthetic pathway combining both terpene cyclase and nonribosomal peptide synthetase enzymes. Flavunoidine consists of a tetracyclic, oxygenated sesquiterpene core decorated with dimethyl cadaverine and 5,5-dimethyl-l-pipecolate moieties. Unique to the flavunoidine biosynthetic pathway is FlvF, a putative enzyme implicated in stereospecific C-N bond formation as dimethyl cadaverine is linked to the sesquiterpene core to generate pre-flavunoidine. Here, we report the 2.6 Å resolution crystal structure of FlvF, which adopts the α-helical fold of a class I terpene synthase. However, FlvF is not a terpene synthase, as indicated by its lack of enzymatic activity with farnesyl diphosphate and its lack of signature metal ion binding motifs that would coordinate to catalytic Mg2+ ions. Thus, FlvF is the first example of a protein that adopts a terpene synthase fold but is not a terpene synthase. Two Bis-Tris molecules bind in the active site of FlvF, and the binding of these ligands guided the docking of pre-flavunoidine to generate a model of the enzyme-product complex. Phylogenetic analysis of FlvF and related fungal homologues reveals conservation of residues that interact with the tetracyclic sesquiterpene in this model, but less conservation of residues interacting with the pendant amino moiety. This may hint toward the possibility that alternative amino substrates can be linked to a common sesquiterpene core by FlvF homologues to generate flavunoidine congeners, such as the phospholipase C inhibitor hispidospermidin.

Hispidospermidin (1) is a novel phospholipase C inhibitor produced by Chaetosphaeronema hispidulum (Cda) Moesz NR 7127. Its structure (C25H47N3O) has been elucidated as a cage compound with a trimethylspermidine side chain based on various NMR studies, including 1H-1H COSY, 13C-1H COSY, HOHAHA, HMBC, COLOC and long range J C-H resolved 2D spectroscopy. The absolute configuration of 1 has been elucidated by modified Mosher's method on the (R)- and (S)-MTPA amides of a derivative of 1.

A novel phospholipase C inhibitor, hispidospermidin, was discovered from a fungal culture broth. The producing fungus, NR 7127, formed abundant pycnidia on banana leaf agar under near UV light. The ostiolate pycnidia were dark colored with a short beak possessing numerous protruding setae. The conidiogeneous cells were phialidic. The conidia were hyaline, 1 septate, smooth and spindle-shaped. From these distinctive characteristics, this strain was identified as Chaetosphaeronema hispidulum (Cda) Moesz of the Coelomycetes. Hispidospermidin was produced in a 50-liter jar fermentor containing 2% glucose, 2% potato starch, 2% Toast soya, 0.5% yeast extract, 0.25% NaCl, 0.0005% ZnSO4.7H2O, 0.0005% CuSO4.5H2O, 0.0005% MnSO4.4H2O, 0.32% CaCO3, and 0.3% Nissan disfoam CA-115. Fermentation was conducted at 27 degrees C at an aeration rate of 30 liters/minute and agitated at 500 rpm for 95 hours. Maximum production yield of hispidospermidin was observed after 72 hours. Hispidospermidin inhibited rat brain phospholipase C at 16 microM of IC50. This is the first recorded discovery of a secondary metabolite from the genus Chaetosphaeronema.