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

Hapalindole U (4) is a validated biosynthetic precursor to ambiguine alkaloids (Angew. Chem. Int. Ed. 2016, 55, 5780), of which biogenetic origin remains unknown. The recent discovery of AmbU4 (or FamC1) protein encoded in the ambiguine biosynthetic pathway (J. Am. Chem. Soc. 2015, 137, 15366), an isomerocyclase that can rearrange and cyclize geranylated indolenine (2) to a previously unknown 12-epi-hapalindole U (3), raised the question whether 3 is a direct precursor to 4 or an artifact arising from the limited in vitro experiments. Here we report a systematic approach that led to the discovery of an unprecedented calcium-dependent AmbU1-AmbU4 enzymatic complex for the selective formation of 4. This 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 a new family of calcium-dependent enzymes, where the metal ions are shown critical for their enzymatic activities and selectivities.