Catalytic mechanism for β-carotene isomerisation and substrate selectivity by the strigolactone biosynthetic enzymes D27 and D27like1

Strigolactones (SLs) are important phytohormones that regulate plant architecture, stress response, and adaptation. SLs exuded by roots also act as signals that allow symbiotic fungi and root-parasitic Striga plants to detect their host. Carlactone, the precursor of SLs, is derived from all-trans-β-carotene through the sequential action of the enzymes DWARF27 (D27), carotenoid cleavage dioxygenase 7 (CCD7), and CCD8. D27 catalyses the isomerisation between all-trans and 9-cis-β-carotene, enriching 9-cis-β-carotene, which is the substrate for CCD7. D27 paralogues (D27likes) have also been reported to use 15-cis- or 13-cis-β-carotene isomers as substrates. The molecular basis for the isomerisation of β-carotene by the D27 enzyme family has remained elusive. By using AI-enabled protein structure prediction to guide experimental and computational methods, we demonstrate that D27 contains a 4Fe–4S cluster positioned deep within a hydrophobic cavity that can accommodate β-carotenes. This configuration allows iron cluster–mediated softening of β-carotene through a single-electron transfer reaction and subsequent cavity-induced stereodivergent isomerisation. Differences in cavity dimensions and stereochemistry explain the differences in isomer preference of D27 and D27like1 proteins. Structure-based analysis proposes that the rim areas lining the catalytic site openings of D27, CCD7, and CCD8 immerse into the membrane, implying a mechanism for sequential substrate capture from the membrane, catalysis, and product release into the membrane. Our findings fill a critical gap in the understanding of SL biosynthesis and may inspire new directed interventions to improve plant growth and resilience.