Hydroxylation with Unusual Stereoinversion Catalyzed by an Fe^II/2‐OG Dependent Oxidase and 3,6‐Diene‐2,5‐diketopiperazine Formation in the Biosynthesis of Brevianamide K

Abstract

Natural products with the 3,6‐diene‐2,5‐diketopiperazine core are widely distributed in nature; however, the biosynthetic mechanism of 3,6‐diene‐2,5‐diketopiperazine in fungi remains to be further elucidated. Through heterologous expression and biochemical investigation of an FeII/2‐oxoglutarate‐dependent oxidase (AspE) and a heme‐dependent P450 enzyme (AspF), we report that AspE, AspF and subsequent dehydration account for the formation of the 3,6‐diene‐2,5‐diketopiperazine substructure of brevianamide K from Aspergillus sp. SK‐28, a symbiotic fungus of mangrove plant Kandelia candel. More interestingly, in‐depth investigation of the enzymatic mechanism showed that AspE promotes hydroxylation of brevianamide Q with unprecedented stereoinversion through hydrogen atom abstraction and water nucleophilic attack from the opposite face of the resultant iminium cation intermediate.

Substrate-Dependent Mechanism Switch in the Desaturation Reactions of the Mononuclear Nonheme Iron Enzyme PtlD

“…Several experimental findings have indicated that the carbocation intermediate proposed in the catalytic mechanism is plausible for the desaturation reactions. For instance, studies on NvfI, TqaL, AspE, KabC, PIsnB, and PvcB have reported such findings. Since the feasibility of single ET between the substrate radical and Fe III –OH [in pathway (c)] is debatable from the thermodynamic perspective, − we should still be cautious regarding the detailed steps in certain cases.…”

“…However, recent studies have reported the occurrence of stereoconfiguration-inversed hydroxylation products in the hydroxylation reaction catalyzed by two NHFe/α-KG enzymes, TqaL and AspE. 19,68 Oxygen incorporation experiments proved that the oxygen atoms of the stereoconfiguration-inversed hydroxyl group originate from H 2 O rather than from O 2 , indicating that a carbocation intermediate is formed and attacked by H 2 O from the opposite face.…”

“…Several experimental findings have indicated that the carbocation intermediate proposed in the catalytic mechanism is plausible for the desaturation reactions. For instance, studies on NvfI, 17 TqaL, 19 AspE, 68 KabC, 70 is debatable from the thermodynamic perspective, 71−73 we should still be cautious regarding the detailed steps in certain cases. The uncertainty may be attributed to factors such as the redox potential of Fe III −OH, the ET barrier between the substrate radical and cation intermediate, the incompatibility of an unstable cation intermediate in the enzyme binding pocket, and the structural properties of substrates.…”

Hot off the Press

“…Biochemical studies have revealed the biosynthetic pathway of brevianamide K 26 , a 3,6-diene-2,5-diketopiperazine from Aspergillus species SK-28. 24 An NRPS catalyses the formation of brevianamide K from l -tryptophan and l -proline, while two oxidative enzymes followed by dehydration are responsible for alkene formation. Gene disruption, feeding experiments and enzymes assays have allowed the elucidation of the biosynthesis of pretrichlodermamide A 27 , a fungal epidithiodiketopiperazine from Trichoderma hypoxylon .…”