Biosynthesis of Spinosyn A: a [4+2] or [6+4] Cycloaddition?

ABSTRACT: SpnF, one of the Diels-Alderases, produces spinosyn A, which has been demonstrated that its sole func-tion is to catalyze the [4+2] cycloaddition (Fage C. D. et al. Nat. Chem. Bio. 2015). Furthermore, the potential existence of a [6+4] cycloaddition bifurcation from previous theoretical cal-culations on the non-enzyme model (Patel A. et al. J. Am. Chem. Soc. 2016), shows that the exact mechanism of SpnF becomes even more interesting as well as now being controversial. Herein QM(DFT)/MM MD simulations on the full-enzyme model reveal three significant residues collaborating with other residues that control the direction of the cycloaddition, namely Tyr23, Thr196 and Trp256. These residues force the substrate into a reactive conformation that causes the cycloaddition reaction to proceed through a [4+2] pathway instead of the [6+4] one. The mechanistic insights deciphered here are fundamentally important for the rational design of Diels-Alderases and biomimetic syntheses.