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.

神经兴奋性红藻氨酸类化合物（neuroexcitatory kainoids）的生物合成，依赖由非血红素铁和2-氧戊二酸依赖型酶（nonheme iron and 2-oxoglutarate-dependent enzymes）催化的L-谷氨酸N-异戊烯基衍生物的自由基介导环化反应。尽管来自红藻物种的KabC与DabC分别在红藻氨酸（kainic acid）和软骨藻酸（domoic acid）的生物合成中催化该反应，但KabC还可生成双环内酯（bicyclic lactone）作为副反应产物。本文系统解析了KabC与DabC以底物N-二甲基烯丙基L-谷氨酸为反应物时的自由基介导催化通路，共明确三类产物决定步骤，以及羟基化、碳碳键形成、分子内亲核加成、去饱和与碳碳键断裂等多种竞争反应过程，最终生成红藻氨酸、双环内酯、羟基化产物及伴随甲醛消除的氧化重排产物共四种不同产物。该反应通过立体选择性夺取底物C3位的前R氢原子启动，随后发生自由基环化，该过程相较于经典的羟基回弹路径更具优势。当使用环丙烷类似物作为底物进行酶活测定时，可观测到开环产物，这为自由基触发的环化过程提供了直接实验证据。针对去饱和与内酯化这两个产物决定步骤测得的一级氘动力学同位素效应均小于2，表明去饱和过程涉及质子耦合电子转移（proton coupled electron transfer, PCET）而非酸碱反应。此外，通过检测到重排产物，也佐证了阳离子中间体的参与。综上，上述观测结果不仅揭示了红藻氨酸类化合物生物合成过程中吡咯烷环形成的复杂性，也证明了该反应的产物结局具有可调控性，这为理解自由基介导酶促反应中不稳定中间体的调控机制提供了重要参考。