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

Structural Elucidation of Cisoid and Transoid Cyclization Pathways of a Sesquiterpene Synthase Using 2-Fluorofarnesyl Diphosphates

Sesquiterpene skeletal complexity in nature originates from the enzyme-catalyzed ionization of (<i>trans</i>,<i>trans</i>)-farnesyl diphosphate (FPP) (<b>1a</b>) and subsequent cyclization along either 2,3-transoid or 2,3-cisoid farnesyl cation pathways. Tobacco 5-epi-aristolochene synthase (TEAS), a transoid synthase, produces cisoid products as a component of its minor product spectrum. To investigate the cryptic cisoid cyclization pathway in TEAS, we employed (<i>cis</i>,<i>trans</i>)-FPP (<b>1b</b>) as an alternative substrate. Strikingly, TEAS was catalytically robust in the enzymatic conversion of (<i>cis</i>,<i>trans</i>)-FPP (<b>1b</b>) to exclusively (≥99.5%) cisoid products. Further, crystallographic characterization of wild-type TEAS and a catalytically promiscuous mutant (M4 TEAS) with 2-fluoro analogues of both all-<i>trans</i> FPP (<b>1a</b>) and (<i>cis</i>,<i>trans</i>)-FPP (<b>1b</b>) revealed binding modes consistent with preorganization of the farnesyl chain. These results provide a structural glimpse into both cisoid and transoid cyclization pathways efficiently templated by a single enzyme active site, consistent with the recently elucidated stereochemistry of the cisoid products. Further, computational studies using density functional theory calculations reveal concerted, highly asynchronous cyclization pathways leading to the major cisoid cyclization products. The implications of these discoveries for expanded sesquiterpene diversity in nature are discussed