The structure of SpnF, a standalone enzyme that catalyzes [4 + 2] cycloaddition

Abstract

In the biosynthetic pathway of the spinosyn insecticides, the tailoring enzyme SpnF performs a [4+2]-cycloaddition on a 22-membered macrolactone to forge an embedded cyclohexene ring. To learn more about this reaction, which could potentially proceed through a Diels-Alder mechanism, the 1.50 Å-resolution crystal structure of SpnF bound to S-adenosylhomocysteine (SAH) was determined. This sets the stage for advanced experimental and computational studies to determine the precise mechanism of SpnF-mediated cyclization.

The Enzymology of Organic Transformations: A Survey of Name Reactions in Biological Systems

“…SpnF structure with the consensus docking of its substrate and product revealed an active cavity forming favorable interactions between SpnF and the macrolactone substrate. [335] However, as all aforementioned [4+2]-cyclases, it remains unclear whether the SpnF reaction proceeds via a pericyclic transition state. Although a concerted, albeit asynchronous, Diels-Alder mechanism for SpnF-catalyzed cyclization is supported by the theoretical study by Hess and Smentek, [336] recent quantum mechanical computations and dynamic simulations show that this cycloaddition is not well described as either a concerted or stepwise process.…”

Natural Diels–Alderases: Elusive and Irresistable

“…[41] While the enzyme of interest resembles S-adenosylmethionine (SAM)-dependent methyltransferases (SAM MTs), the activity of SAM MTs could give insight to the activity of the stand-alone enzyme. The activity of SAM MTs in these particular cyclization reactions is currently unknown.…”

“…The activity of SAM MTs in these particular cyclization reactions is currently unknown. [41a] The authors conclude however, that: “The mechanisms by which SpnF and SpnL catalyse their respective cyclization reactions are a point of interest. The SpnF-catalysed endo mode syn-addition of an alkenyl to a dienyl functionality seems consistent with a Diels–Alder reaction; however, confirmation of this hypothesis will require demonstrating that the reaction progresses through a single pericyclic transition state such as [ 6 ].…”

A chemocentric view of the natural product inventory

“…(i) Over the past decade the purported Diels-Alderases have been put on a much stronger mechanistic and structural footing, in part due to the reported X-ray structure of SpnF (ref. 7). This enzyme carries out a [4+2] cyclization of a biosynthetic precursor to yield a cyclohexene embedded as a fused 5/6 bicycle within the 22-membered spinosyn macrolactone framework (Fig.…”