Biochemical Characterization of NotB as an FAD-Dependent Oxidase in the Biosynthesis of Notoamide Indole Alkaloids

Abstract

Notoamides produced by Aspergillus spp. bearing the bicyclo [2.2.2] diazaoctane core structure with unusual structural diversity represent a compelling system to understand the biosynthesis of fungal prenylated indole alkaloids. Herein, we report the in vitro characterization of NotB, which catalyzes the indole 2,3-oxidation of notoamide E (13), leading to notoamide C (11) and D (12) through an apparent Pinacol-like rearrangement. This unique enzymatic reaction with high substrate specificity, together with the information derived from the precursor incorporation experiments using [13C]2-[15N]2 quadruply labeled notoamide S (10) demonstrates 10 as a pivotal branching point in notoamide biosynthesis.

Structural and stereochemical diversity in prenylated indole alkaloids containing the bicyclo[2.2.2]diazaoctane ring system from marine and terrestrial fungi

“…7). 77 These results clearly indicated that the construction of the bicyclo[2.2.2]diazaoctane ring system occurs prior to the formation of the pyran ring. Although notoamides S and T had not previously been found in the culture extract of A. amoenus , Kato, et al ., have recently identified notoamide S as a minor metabolite of A. amoenus .…”

“…77 Curiously, 3- epi -notoamide C is not observed as a product when notoamide E is exposed to recombinant NotB. 3- epi -Notoamide C has been previously isolated from a precursor incorporation experiment with notoamide E as an added substrate, but has not been directly isolated from fermentation extracts of Aspergillus protuberus .…”

A Semipinacol Rearrangement Directed by an Enzymatic System Featuring Dual‐Function FAD‐Dependent Monooxygenase

“…[7,8] Pinacol rearrangements are proposed to occur during the biosyntheses of various secondary metabolites, including aflatoxin B1, (+)-liphagal, (+)-asteltoxin, brevianamides, paraherquamide B, verscicolamide B, and notoamides. [9][10][11] However, no such biosynthetic hypotheses has been biochemically proven, although the proposed pathways in turn inspired biomimetic approaches for natural product synthesis. [9] Therefore, an enzymatic system for pinacol-type rearrangement remained to be discovered in the biosynthesis of secondary metabolites.…”

Biosynthesis of fungal indole alkaloids

“…The hydroxyiminium species is electrophilic and can be readily attacked at C2 by a nucleophile and form a C3-hydroxylated indoline. The nucleophilic attack can be frequently intramolecular, such as from the amide nitrogen as in the case during notoamide D biosynthesis, 29 or be intermolecular, as from a free amino group during biosynthesis of 7 . 13 Interestingly, the presence of a C6 O -substitution in the benzyl ring can direct the 2, 3-epoxide intermediate to open at C3 position and undergo a semipinacol-like rearrangement, which results in the migration of the C2-substitution to C3 position to form a substituted 2-indolone, as in biosynthesis of notoamide C. 29 ( Scheme 2B )…”