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

SpnF, one of the Diels–Alderases, produces spinosyn A, and previous work demonstrated that its sole function is to catalyze the [4 + 2] cycloaddition (Fage, C. D.; et al. Nat. Chem. Biol. 2015, 11, 256−258). Furthermore, the potential existence of a [6 + 4] cycloaddition bifurcation from previous theoretical calculations on the nonenzyme model (Patel, A.; et al. J. Am. Chem. Soc. 2016, 138, 3631−3634) shows that the exact mechanism of SpnF becomes even more interesting as well as now being controversial. In the present work, QM­(DFT)/MM MD simulations on the full enzyme model revealed three significant residues that collaborate with other residues to 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.