Decoding cyclase-dependent assembly of hapalindole and fischerindole alkaloids

Abstract

The formation of C-C bonds in an enantioselective fashion to create complex polycyclic scaffolds in the hapalindole/fischerindole type alkaloids from Stigonematales cyanobacteria represents a compelling and urgent challenge in adapting microbial biosynthesis as a catalytic platform in drug development. Here we determine the biochemical basis for tri- and tetracyclic core formation in these secondary metabolites, involving a new class of cyclases that catalyze a complex cyclization cascade.

Structural basis of the Cope rearrangement and C–C bond-forming cascade in hapalindole/fischerindole biogenesis

“…Following recent work that reported the function and selectivity of Stig cyclase proteins (10,(14)(15)(16), we sought a crystal structure of HpiC1 in an effort to understand the structural basis of the complex cyclization cascade of its primary substrate 1, and to understand how the Stig cyclases catalyze formation of variant alkaloid products from this common biosynthetic intermediate. Ultimately, we obtained HpiC1 crystals in four different forms, under conditions that differed in Ca 2+ concentration.…”

“…transformed to products in a regio-and stereospecific fashion ( Fig. 1b) (14)(15)(16). Thus, biogenesis of hapalindole-type metabolites includes a fascinating mechanistic puzzle regarding how homologous Stig cyclases mediate formation of varied hapalindoles or fischerindoles based on a remarkable C-C bond forming cascade.…”

“…1b). This three-part reaction scheme is employed by the varied Stig cyclases which maintain exquisite stereochemical and regiochemical control through each of these mechanistic steps (14)(15)(16).…”

Discovery of a Calcium‐Dependent Enzymatic Cascade for the Selective Assembly of Hapalindole‐Type Alkaloids: On the Biosynthetic Origin of Hapalindole U

“…Thei dentification of the 10xAmbU1-U4 enzymatic complex as the dedicated hapalindole Usynthase also allowed to establish 4 as the bona fide biosynthetic intermediate for the assembly of hapalindole A, the founding member of hapalindole-type alkaloids, [18] based on the co-occurrence of AmbU1-AmbU4 coding genes in its biosynthetic gene cluster sequenced from Fischerella muscicola UTEX LB1829 ( Figure S20). [21] In addition, the disclosure on the in vitro enzymatic assembly of 4,a no n-pathway biogenetic intermediate to ambiguines and related hapalindole alkaloids,t hat requires a1 0-to-1 mixture of AmbU1 and AmbU4 in the presence of calcium contrasts ar ecent claim, emerged during the preparation of this manuscript, [22] that states the structural diversifications from 2 in the biosynthesis of hapalindoletype alkaloids is controlled by the homo-or hetero-dimeric complex of the U-type isomerocyclases.W er easoned this over-interpretation is likely due to the failure to appreciate that 6 is apathway outlier in the biogenesis of ambiguines and related hapalindole alkaloids.A st he in vitro enzymatic reconstitution remains the only viable method to dissect the biochemical basis for the biosynthesis of hapalindole-type alkaloids,w ec aution for the pitfalls in the interpretation of these in vitro data without thoroughly correlating them with the observed structural diversity in the known hapalindoletype alkaloid producer. [21] In addition, the disclosure on the in vitro enzymatic assembly of 4,a no n-pathway biogenetic intermediate to ambiguines and related hapalindole alkaloids,t hat requires a1 0-to-1 mixture of AmbU1 and AmbU4 in the presence of calcium contrasts ar ecent claim, emerged during the preparation of this manuscript, [22] that states the structural diversifications from 2 in the biosynthesis of hapalindoletype alkaloids is controlled by the homo-or hetero-dimeric complex of the U-type isomerocyclases.W er easoned this over-interpretation is likely due to the failure to appreciate that 6 is apathway outlier in the biogenesis of ambiguines and related hapalindole alkaloids.A st he in vitro enzymatic reconstitution remains the only viable method to dissect the biochemical basis for the biosynthesis of hapalindole-type alkaloids,w ec aution for the pitfalls in the interpretation of these in vitro data without thoroughly correlating them with the observed structural diversity in the known hapalindoletype alkaloid producer.…”

“…[19,20] This corroborates with our recent observation that an enzyme-mediated tertiary carbon epimerization at the C-10 center of 4 and 5 is operant for the stereodivergent biogenesis of the hapalindoletype alkaloids in the same organism. [21] In addition, the disclosure on the in vitro enzymatic assembly of 4,a no n-pathway biogenetic intermediate to ambiguines and related hapalindole alkaloids,t hat requires a1 0-to-1 mixture of AmbU1 and AmbU4 in the presence of calcium contrasts ar ecent claim, emerged during the preparation of this manuscript, [22] that states the structural diversifications from 2 in the biosynthesis of hapalindoletype alkaloids is controlled by the homo-or hetero-dimeric complex of the U-type isomerocyclases.W er easoned this over-interpretation is likely due to the failure to appreciate that 6 is apathway outlier in the biogenesis of ambiguines and related hapalindole alkaloids.A st he in vitro enzymatic reconstitution remains the only viable method to dissect the biochemical basis for the biosynthesis of hapalindole-type alkaloids,w ec aution for the pitfalls in the interpretation of these in vitro data without thoroughly correlating them with the observed structural diversity in the known hapalindoletype alkaloid producer.…”