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, we determined the 1.50-Å-resolution crystal structure of SpnF bound to S-adenosylhomocysteine. This sets the stage for advanced experimental and computational studies to determine the precise mechanism of SpnF-mediated cyclization.

Figure 1

Reactions mediated by SpnF and accompanying tailoring enzymes. The processive spinosyn Type I PKS generates spilactone, which is processed by several tailoring enzymes into spinosad (Spinosyns A and D). SpnF performs a [4+2]-cyclization, possibly through a Diels-Alder mechanism, between the C4–C7 diene (C5–C6 bond in s-cis geometry) and the C11–C12 dienophile, embedding a cyclohexene ring in the product (conformations minimized with the MMFF94s force field).

Figure 2

SpnF structure and consensus docking of its substrate and product. a) SpnF adopts a SAM-dependent methyltransferase (MT) fold, with a catalytic core and a cover like most natural product MTs. The electron density maps reveal the presence of two small molecules bound to each SpnF monomer – SAH and what appears to be malonate (MLI) in two conformations. The F_o−F_c omit map, contoured at 4.0 r.m.s.d., shows SAH from one of the two SpnF monomers in the asymmetric unit. b) The programs AutoDock Vina and DOCK6 were used to dock SpnF substrate and product into the substrate cavity (structures minimized with the MMFF94s force field are shown here; see Supplementary Table 2 for more detail). A top orientation for both molecules in both programs is shown. The majority of contacts are hydrophobic although the C17-OH forms hydrogen bonds with both H42 and Q148. The C11–C12 π-bond may be polarized by T196 through its hydrogen bond to the C15 keto group to react with the C4–C7 diene. SAH does not make contact with either the substrate or product.

Figure 3

Cyclization assays of SpnF mutants. Point mutants were generated and compared to wild-type SpnF through a cyclization assay with triplicate measurements (data represent mean values ± s.d.). That Q148E showed the greatest loss in activity may be attributed to the Q148 side chain amino group making contact with the SpnF substrate during catalysis.