Multiple Catalytic Branch Points in the Mechanism of Pyrrolidine Formation During Kainoid Biosynthesis Leads to Diverse Reaction Outcomes

The biosynthesis of neuroexcitatory kainoids requires radical-mediated cyclization of N-isoprenylated derivatives of l-glutamate catalyzed by nonheme iron and 2-oxoglutarate-dependent enzymes. While KabC and DabC from species of red algae catalyze this reaction during the biosynthesis of kainic acid and domoic acid, respectively, KabC can also produce a bicyclic lactone as an alternative reaction product. Herein, the radical-mediated catalytic pathways of KabC and DabC with the substrate N-dimethylallyl l-glutamate are fully mapped demonstrating as many as three different product determining steps and competing processes of hydroxylation, C–C bond formation, intramolecular nucleophilic addition, desaturation and C–C bond cleavage leading to four different products including kainic acid, a bicyclic lactone, a hydroxylated product and oxidative rearrangement concomitant with elimination of formaldehyde. The reaction proceeds via stereoselective abstraction of the pro-R H atom from C3 of the substrate followed by radical cyclization that outcompetes canonical hydroxy rebound. Evidence of radical triggered cyclization is provided by the observation of a ring-opened product when a cyclopropyl analogue is assayed. Measurement of primary deuterium kinetic isotope effects less than 2 on the product determining step of desaturation versus lactonization suggests the former involves proton coupled electron transfer (PCET) rather than an acid-base reaction. Furthermore, involvement of a cationic species is supported by detection of a rearrangement product. Collectively, these observations not only reveal the complexity of pyrrolidine formation during kainoid biosynthesis but also its amenability to changes in reaction outcome, which is of use for understanding the control of unstable intermediates during radical-mediated enzymatic reactions.