Oxepinamide F biosynthesis involves enzymatic d-aminoacyl epimerization, 3H-oxepin formation, and hydroxylation induced double bond migration

Abstract

Oxepinamides are derivatives of anthranilyl-containing tripeptides and share an oxepin ring and a fused pyrimidinone moiety. To the best of our knowledge, no studies have been reported on the elucidation of an oxepinamide biosynthetic pathway and conversion of a quinazolinone to a pyrimidinone-fused 1H-oxepin framework by a cytochrome P450 enzyme in fungal natural product biosynthesis. Here we report the isolation of oxepinamide F from Aspergillus ustus and identification of its biosynthetic pathway by gene deletion, heterologous expression, feeding experiments, and enzyme assays. The nonribosomal peptide synthase (NRPS) OpaA assembles the quinazolinone core with d-Phe incorporation. The cytochrome P450 enzyme OpaB catalyzes alone the oxepin ring formation. The flavoenzyme OpaC installs subsequently one hydroxyl group at the oxepin ring, accompanied by double bond migration. The epimerase OpaE changes the d-Phe residue back to l-form, which is essential for the final methylation by OpaF.

Oxepin Formation in Fungi Implies Specific and Stereoselective Ring Expansion

“…As reported previously, OpaB from the oxapinamide F BGC catalyzed the formation of the oxepin ring in the OPK backbone. 8 Although OpaB2 shares only a sequence identity of 30% with OpaB, it could still be responsible for oxepin ring formation in oxepinamide D. Deletion of opaB2 from the A. ustus genome led indeed to the abolishment of 4 and the accumulation of two tripeptide derivatives, 5 and 6 (Figure 2iv), the same products after opaA2 expression in A. nidulans (Figure 3i). Again, 6 was detected as the predominant product.…”

“…To determine whether this change in configuration is essential for the oxepin ring formation catalyzed by OpaB, (14S)-epi-protuboxepin K (9) was chemically synthesized according to the methods reported previously 14,15 and fed into the culture of an A. nidulans opaB transformant created in the previous study. 8 As shown in Figure 4A, no product was detected by HPLC analysis. In contrast, almost complete conversion of the natural substrate protuboxepin K (8) to protuboxepin A (10) was observed after feeding into the A. nidulans opaB culture.…”

“…Despite the structural diversity of OPKs, only one biosynthetic pathway, i.e., that of oxepinamide F from Aspergillus ustus, has been recently reported by our group. 8 A. usuts 3.3904 is a rare pathogenic fungus and produces different secondary metabolites, e.g., ustethylin A (1) 9 and oxepinamides E (2) and F (3) (Figures 1B and 2i). In this study, we identified an additional OPK derivative, oxepinamide D [4 (Figures 1B and 2i)], in A. usuts 3.3904.…”

“…Since 1988, more than 37 OPK derivatives including oxepinamides were identified. , Interestingly, oxepinamides consisting of different amino acids were often isolated from the same strains. , This phenomenon raised the question if they are products of one or more biosynthetic gene clusters (BGCs). Despite the structural diversity of OPKs, only one biosynthetic pathway, i.e., that of oxepinamide F from Aspergillus ustus , has been recently reported by our group …”

“…A. usuts 3.3904 is a rare pathogenic fungus and produces different secondary metabolites, e.g., ustethylin A ( 1 ) and oxepinamides E ( 2 ) and F ( 3 ) (Figures B and i) . Oxepinamide F is an O-methylated product of oxepinamide E. In their biosynthetic pathway, the NRPS OpaA with a domain architecture of A-T-C-A-T-E-C-A-T-C (A, adenylation; T, thiolation; C, condensation; E, epimerization) utilizes Ant, Phe, and Ile to assemble a fused quinazolinone derivative.…”