Recognizing that rice produced numerous labdane-related diterpenoid natural products, and the opportunity presented by the publication of the rice genome sequence, we began by carrying out a functional genomics based investigation of the rice diterpene synthases. Given the relative paucity of characterized diterpene synthases at that time, this effort more than doubled the number of molecularly identified functionally distinct such enzymes. Strikingly, we further uncovered a functionally distinct pair of alleles for one of the rice class I diterpene synthases, which led to isolation of a single residue ‘switch’ for product outcome. The identity of the residue at this position has a profound effect on the catalyzed reaction, with the presence of an aliphatic side-chain counter-intuitively leading to a more complex reaction, while a hydroxyl containing side-chain leads to termination at an earlier step (i.e., following initial cyclization). Our results provide support for the hypothesis that the electrophilic reactions catalyzed by (di)terpene synthases, with highly-reactive carbocationic intermediates, are under kinetic rather than thermodynamic control, as well as a role for the pyrophosphate co-product as a counter-ion that drives carbocation migration towards itself. Our mechanistic hypothesis for the observed effects is that hydroxyl dipole stabilization of the specific carbocation formed by initial cyclization enables deprotonation, whereas the lack of such stabilization (i.e. in the presence of an aliphatic side chain) leads to carbocation migration towards the pyrophosphate co-product, resulting in a more complex reaction. Thus, (di)terpene synthases seem to mediate specific product outcomes, at least in part, by providing electrostatic effects to counteract those exerted by the pyrophosphate co-product.
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