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

Abstract

Hapalindole U( 4)i savalidated biosynthetic precursor to ambiguine alkaloids (Angew.C hem. Int. Ed. 2016,55, 5780), of whichbiogenetic origin remains unknown. The recent discovery of AmbU4 (or FamC1) protein encoded in the ambiguine biosynthetic pathway (J.A m. Chem. Soc. 2015,1 37, 15366), an isomerocyclase that can rearrange and cyclizegeranylated indolenine (2)toapreviously unknown 12epi-hapalindole U(3), raised the question whether 3 is adirect precursor to 4 or an artifact arising from the limited in vitro experiments.Here we report asystematic approachthat led to the discovery of an unprecedented calcium-dependent AmbU1-AmbU4 enzymatic complex for the selective formation of 4.T his discovery refuted the intermediacy of 3 and bridged the missing links in the early-stage biosynthesis of ambiguines.This work further established the isomerocyclases involved in the biogenesis of hapalindole-type alkaloids as an ew family of calcium-dependent enzymes,w here the metal ions are shown critical for their enzymatic activities and selectivities.

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

“…4). With some cyclases, a requirement for millimolar concentrations of Ca 2+ in the reaction buffer to achieve activity has been reported (15). Taken together these data are indicative of an important, yet complex structural role for Ca 2+ in the Stig cyclases.…”

“…These sites were a key starting point to assess the structural and catalytic role that Ca 2+ plays in the Stig cyclase enzymes. In vitro assays conducted in the presence of 5 mM EDTA showed no activity, leading us to conclude that Ca 2+ is required for catalytic function in HpiC1 (15). From sequence comparisons, we expect the integral calcium-binding sites and the core dimeric assembly to be maintained in the other Stig cyclase enzymes ( Fig.…”

“…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).…”