Dirigent Proteins from Cotton (Gossypium sp.) for the Atropselective Synthesis of Gossypol

Abstract

Gossypol is a defense compound in cotton plants for protection against pests and pathogens. Gossypol biosynthesis involves the oxidative coupling of hemigossypol and results in two atropisomers owing to hindered rotation around the central binaphthyl bond. (+)-Gossypol predominates in vivo, thus suggesting stereochemically controlled biosynthesis. The aim was to identify the factors mediating (+)-gossypol formation in cotton and to investigate their potential for asymmetric biaryl synthesis. A dirigent protein from Gossypium hirsutum (GhDIR4) was found to confer atropselectivity to the coupling of hemigossypol in presence of laccase and O2 as an oxidizing agent. (+)-Gossypol was obtained in greater than 80% enantiomeric excess compared to racemic gossypol in the absence of GhDIR4. The identification of GhDIR4 highlights a broader role for DIRs in plant secondary metabolism and may eventually lead to the development of DIRs as tools for the synthesis of axially chiral binaphthyls.

The Missing Link in Leguminous Pterocarpan Biosynthesis is a Dirigent Domain-Containing Protein with Isoflavanol Dehydratase Activity

“…Consistent with this, the recombinant GhDIR4 was detected in a truncated form in cultured tomato cells (Effenberger et al 2015). Thus, for biochemical analysis of GePTS1, GmPTS1 and LjPTS1, recombinant proteins were expressed as signal peptide-truncated and histidine-tagged forms in E. coli , and their enzymatic properties were examined after affinity purification.…”

“…The amino acid sequences were found to include the characteristic motif (Ralph et al 2007) originally reported in DIR, which governs stereospecific phenoxy radical coupling in collaboration with an oxidase to form enantiopure (+)-pinoresinol during lignan biosynthesis (Davin et al 1997). The molecular characteristics of other DIRs have since been investigated (Pickel and Schaller 2013), and some are involved in the formation of the lignin-based Casparian strip in roots, while others play a role in cotton phytoalexin biosynthesis (Hosmani et al 2013, Effenberger et al 2015). Before the present study, it was believed that DIR did not possess enzymatic activity, and simply regulated product configuration.…”

Dirigent Protein Mode of Action Revealed by the Crystal Structure of AtDIR6

“…AtDIR6 mutants and the wild-type control were transiently expressed in Nicotiana benthamiana by agroinfiltration as described previously for cotton (Gossypium spp.) DIRs (Effenberger et al, 2015). The leaves were harvested after 3 d, cut into pieces of 3 to 5 cm 2 , and rinsed in ice-cold deionized water.…”

“…Moreover, we recently found a novel type of DIR in cotton (Gossypium spp.) conferring atropselectivity to the oxidative coupling of hemigossypol (Effenberger et al, 2015). This reaction constitutes the final step in the biosynthesis of the sesquiterpenoid dimer gossypol, an important natural product and defense compound in cotton plants (Wagner et al, 2012;Tian et al, 2016).…”

Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease

“…Proteins were transiently expressed in N. benthamiana by co-infiltration of two Agrobacterium tumefaciens strains, one carrying the pART27 expression vector, the other carrying the p19 suppressor of silencing essentially as described (58). The bacterial suspension was infiltrated into the abaxial side of leaves from 6-week-old N. benthamiana plants.…”