Insights into the Multisymmetric Tailoring of Julichrome Compounds by the Oxygenases JuiN and JuiO: Enzymatic Processing around the Biaryl C–C Axis

Biaryl moieties, especially in the context of natural products (NPs), constitute a privileged scaffold for drug design. However, compound modifications that follow the installation of the defining C–C biaryl axis have not been well documented. Here, we report two iterative monooxygenases, JuiN and JuiO, that catalyze tandem and symmetric hydroxylation and epoxidation chemistries following C–C axis formation en route to the NP julichrome Q6-6. The crystal structure of JuiN-FAD-Q6-6 and mutagenesis experiments revealed the substrate recognition mechanisms driving iterative hydroxylations along the C–C axis. The key to these studies was that the unstable intermediate julichrome Q8-8 was successfully captured using an adsorptive resin in fermentations of a ΔjuiO mutant, thus invoking JuiO in the overall sequence of transformations. A Streptomyces griseorubiginosus-derived enzyme, SgJuiO, was investigated in vitro as a substitute for insoluble JuiO. The biochemical reactions unambiguously demonstrated that SgJuiO iteratively catalyzes the complex sequence of epoxidations on both sides of the C–C biaryl linkage. These findings showcase a tailoring paradigm for biaryl NP biosynthesis and engineering.