Comparative analysis of the biosynthetic systems for fungal bicyclo[2.2.2]diazaoctane indole alkaloids: the (+)/(−)-notoamide, paraherquamide and malbrancheamide pathways

Abstract

The biosynthesis of fungal bicyclo[2.2.2]diazaoctane indole alkaloids with a wide spectrum of biological activities have attracted increasing interest. Their intriguing mode of assembly has long been proposed to feature a non-ribosomal peptide synthetase, a presumed intramolecular Diels-Alderase, a variant number of prenyltransferases, and a series of oxidases responsible for the diverse tailoring modifications of their cyclodipeptide-based structural core. Until recently, the details of these biosynthetic pathways have remained largely unknown due to lack of information on the fungal derived biosynthetic gene clusters. Herein, we report a comparative analysis of four natural product metabolic systems of a select group of bicyclo[2.2.2]diazaoctane indole alkaloids including (+)/(−)-notoamide, paraherquamide and malbrancheamide, in which we propose an enzyme for each step in the biosynthetic pathway based on deep annotation and on-going biochemical studies.

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

“…This is illustrated in Scheme 31. 62 Thus, Li, et al ., have devised a new biogenetic hypothesis that envisions reductive release of the pro–trp dipeptide from the NRPS module, by either a two-electron or four-electron process (the four-electron reduction is shown) providing species 131 (or, the corresponding aldehyde if the reductive release is a two-electron process) which is then reverse-prenylated and oxidized (for 131 ; or just reverse-prenylated for the two-electron process) to give key amino-aldehyde intermediate 132 that should spontaneously cyclize to carbinolamine 133 . Subsequent dehydration to 134 and tautomerization to azadiene species 135 would then allow for IMDA cycloaddition, directly providing pre-paraherquamide (R = Me) and pre-malbrancheamide (R = H), respectively.…”

“…64 It is reasonable to assume that the initial oxidation of the 2,3-disubstituted indole of 6- epi -notoamide T is conducted by NotI that Li, et al ., previously predicted this oxidation and spiro-rearrangement. 62 The downstream transformations from 6- epi -notoamide T9, requires a minimum of six enzyme-catalyzed reactions: (1) aromatic hydroxylase of T9 to 19 ; (2) a catechol oxidase to 20 ; (3) glycine aminotransferase to 21 ; (4) decarboxylase/cyclase to 23 ; (5) oxazolidine oxidase to T3; and (6) oxazolidinone oxidase to T4. The complex array of transformations required to construct T3 and T4 from 6- epi -notoamide T mandates that multiple dormant tailoring genes extant in this organism were activated by the presence of one (or both) enantiomers of 6- epi -notoamide T. Significant effort will need to be devoted to elucidating the nature of these remarkable transformations that have significant implications for the plasticity of secondary metabolomes in Nature.…”

“…62,83 Table 1 provides a summary of the relevant data on each individual gene and the heterologous host in which the protein was functionally expressed.…”

“…NotG appears to be a cytochrome P450 oxidase responsible for the transformation of deoxybrevianamide E into 6-hydroxydeoxybrevianamide E, which was deduced through a process of elimination by comparative analysis with several fungal gene clusters. 62,83 The other cytochrome P450, NotH is the current most plausible candidate for the oxidative transformation of notoamide S into notoamide T and 6- epi -notoamide T. 83 Much to our chagrin, NotH and NotH´, which is currently the focus of where the key enantiodivergence occurs, is a membrane-associated cytochrome P450 that has proven recalcitrant to functional expression in a heterologous host and constitutes the focus of our current efforts.…”

“…8) revealing that 6- epi -notoamide T is the biosynthetic precursor to versicolamide B and 6- epi -stephacidin A and provided the substrates from which the hitherto unobserved new metabolites were constructed from dormant tailoring genes. 62–64 Efforts are underway to prepare optically pure samples of the individual enantiomers of 6- epi -notoamide T to more deeply interrogate the triggering mechanism for the expression of the dormant tailoring genes responsible for the biosynthesis of these new structures which were only produced in the presence of synthetic, racemic 6- epi -notoamide T.…”