P450-Mediated Dual Cyclization Mechanisms for Pyrroloindoline Unit Formation in Bispyrrolidinoindoline Diketopiperazine Alkaloid Biosynthesis

Bispyrrolidinoindoline-diketopiperazines (BPI-DKP) alkaloids, characterized by a complex 3a,3a'-bispyrrolidino[2,3-<i>b</i>]indoline scaffold, constitute a large class of synthetically challenging bioactive pyrroloindoline natural products. Their biosynthesis, mediated by P450 enzymes, involves oxidative dimerization of diketopiperazines (DKPs), yet the underlying mechanistic and stereochemical details have remained unclear. Here, we elucidate the molecular mechanism of TtpB1, a P450 dimerase that stereoselectively couples two DKP units to form <i>C</i>₂-symmetric BPI scaffolds through an unexpected cascade. Contrary to the prevailing double-radical coupling hypothesis, integrated structural, biochemical, and computational studies reveal a stepwise process: (i) Compound I-mediated generation of a nitrogen-centered DKP radical, triggering cyclization to a C3-radical pyrroloindoline intermediate; (ii) radical addition to the C3' position of a second DKP; and (iii) single-electron transfer (SET)-driven cyclization to construct the second pyrroloindoline unit. This dual N-centered radical- and SET-driven cyclization showcases unprecedented versatility in natural product dimer biosynthesis. Molecular dynamics and QM/MM calculations further demonstrate how substrate conformational dynamics enforce stereochemical fidelity, yielding the <i>C</i>₂-symmetric BPI-DKP core with vicinal quaternary stereocenters. These findings provide the first structure-guided mechanistic elucidation of BPI-DKP synthase, laying the groundwork for rational enzyme engineering and biomimetic synthesis of these pharmacologically important alkaloids.