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.