Characterization of the Biosynthesis of Pimprinine-Type Indolyloxazoles Unravels an Unusual <scp>d</scp>-Configurational Substrate Metabolic Streamline

Pimprinine-type indolyloxazole alkaloids (PIAs), originally discovered in <i>Streptomyces</i>, show a diverse range of important pharmaceutical and agricultural bioactivities, yet their biosynthesis remains unknown. Herein, we report the identification of the biosynthetic enzymes responsible for the formation of the indolyloxazole structure of PIAs from a rhizosphere-derived <i>Streptomyces netropsis</i> NZY3, which involves three key enzymes, PimA (GCN5-related <i>N</i>-acetyltransferase, GNAT), PimB (pyridoxal 5'-phosphate-dependent tryptophan racemase), and PimC (Fe^II/α-ketoglutarate-dependent dioxygenase, Fe^II/αKGD), notably by an unprecedented d-configurational substrate metabolic streamline. First, PimB acts as a gatekeeper to donate the d-tryptophan precursor for the PIA pathway from the l-tryptophan pool of primary metabolism. Subsequently, a unique d-tryptophan GNAT, PimA, catalyzes the formation of <i>N</i>-acyl d-tryptophan. Finally, another novel <i>N</i>-acyl d-tryptophan-specific Fe^II/αKGD, PimC, finishes the formation of an indolyloxazole structure through a proposed radical rearrangement-mediated ring closure mechanism, which is supported by a series of deuterium- and ¹⁸O-labeling experiments <i>in vitro</i>. PimC also catalyzes the formation of the <i>trans</i>-vinyl group containing shunt products <b>1a</b> to <b>3a</b> through an oxygen-rebound mechanism followed by dehydration and decarboxylation or a carbocation-involved decarboxylation pathway. Furthermore, comparative genomic mining reveals that PIA biosynthetic gene clusters (PIAs BGCs) are widely distributed in <i>Actinobacteria</i> and <i>Myxobacteria</i>, suggesting the potential for discovering new PIA-producing strains. This work expands our knowledge about the biosynthetic mechanisms of pharmaceutic-valued indolyloxazole alkaloids, laying an important foundation for their future production through synthetic biology and metabolic engineering strategies.