Cofactor-Independent Pinacolase Directs Non-Diels-Alderase Biogenesis of the Brevianamides

Abstract

<p>Fungal bicyclo[2.2.2]diazaoctane indole alkaloids demonstrate intriguing structures and a wide spectrum of biological activities. Although biomimetic total syntheses have been completed for representative compounds of this structural family, the details of their biogenesis have remained largely uncharacterized. Among them, Brevianamide A represents the most basic form within this class bearing a dioxopiperazine core structure and a rare 3-<i>spiro</i>-<i>psi</i>-indoxyl skeleton. Here, we identified the Brevianamide A biosynthetic gene cluster from <i>Penicillium brevicompacticum</i> NRRL 864 and fully elucidated the metabolic pathway by targeted gene disruption, heterologous expression, precursor incorporation studies, and <i>in vitro</i> biochemical analysis. In particular, we determined that BvnE is a cofactor-independent isomerase that is essential for selective production of Brevianamide A. Based on a high resolution crystal structure of BvnE, molecular modeling, mutational analysis, and computational studies provided new mechanistic insights into the diastereoselective formation of the 3-<i>spiro</i>-<i>psi</i>-indoxyl moiety in Brevianamide A. This occurs through a biocatalyst controlled semi-Pinacol rearrangement and a subsequent spontaneous intramolecular [4+2] <i>hetero</i>-Diels-Alder cycloaddition.</p>

Enzyme evolution in fungal indole alkaloid biosynthesis

“…7B) [50]. Therefore, the preponderance of 17 over 18 is likely due to intrinsic energy barriers in the cycloaddition reaction (nonenzyme catalyzed) [52]. Through the utilization of frontier molecular orbital theory, it was predicted that, for the energy levels of a relatively electron‐rich diene (such as that in the DKP) to effectively interact with a dienophile, powerful electron‐withdrawing groups would need to be present in the dienophile.…”

“…Since the prenyl group is an isolated electron‐neutral vinyl group, one would not expect the [4 + 2] cycloaddition in DKPs to be spontaneous without catalysis, but biomimetic syntheses [53] have shown that the azadiene undergoes a spontaneous IMDA to generate a mixture of brevianamides with enantiomorphic bicyclo[2.2.2] ring systems. While spontaneous, diastereocontrol of the IMDA may be impacted by upstream transformations such as the early‐stage semipinacol rearrangement to generate the 3‐spiro‐ψ‐indoxyl species [52].…”

“…An alternative mechanism for the formation of the bicyclo[2.2.2]diazaoctane ring was discovered for the biosynthesis of brevianamide A [52]. While the previously discussed MKP pathways involved formation of the bicyclic core prior to spirocycle formation, brevianamide production employs a reversed sequence of biosynthetic steps where the oxidized indole intermediate is formed prior to a subsequent DKP oxidation and cyclization.…”

“…This can either cyclize to form brevianamide E ( 45 ) or undergo a semipinacol rearrangement to form an indoxyl intermediate. Oxidation to the azadiene would provide the proper substrate for the IMDA [50,52]. The semipinacol rearrangement is proposed to proceed through general base catalysis to generate the indoxyl [52].…”

“…Oxidation to the azadiene would provide the proper substrate for the IMDA [50,52]. The semipinacol rearrangement is proposed to proceed through general base catalysis to generate the indoxyl [52]. In comparison, paraherquamide biosynthesis utilizes general acid catalysis to generate an oxindole moiety [61], demonstrating that these systems have evolved divergent mechanisms for spirocycle formation.…”