CYP76B74 catalyzes the 3''-hydroxylation of geranylhydroquinone in shikonin biosynthesis

Shikonin and its derivatives are the most abundant naphthoquinone pigments formed in species of the medicinally and economically valuable Boraginaceae. A key step in the shikonin biosynthetic pathway, namely the C-399 hydroxylation of prenylated phenolic intermediate geranylhydroquinone, is expected to be catalyzed by a cytochrome P450. To find cytochrome P450 candidates with transcription profiles similar to those of the known upstream genes, we carried out co-expression analysis of transcriptome datasets of shikonin-proficient and shikonin-deficient cell lines and spatial expression in different organs of Arnebia euchroma. Using geranylhydroquinone as the substrate, biochemical assays demonstrated that CYP76B74 exhibited geranylhydroquinone 399-hydroxylase activity to produce 399-hydroxy-geranylhydroquinone. In the CYP76B74 RNAi A. euchroma hairy roots, shikonin derivative accumulation decreased dramatically, which demonstrated the CYP76B74 requirement for shikonin biosynthesis in the plant. Phylogenetic analysis confirmed that CYP76B74 belonged to the CYP76B subfamily and most likely derived from a more ancestral geraniol 10-hydroxylase. Subcellular localization of CYP76B74 N-terminal reporter fusion directed it to the endoplasmic reticulum membranes. Our results demonstrate that CYP76B74 catalyzes the key hydroxylation step in shikonin biosynthesis with high efficiency. The characterization of the CYP76B74 described here paved the way for further exploration of the ring closure reactions generating naphthoquinone skeleton, as well as for the alternative metabolization of geranylhydroquinone 399-hydroxylase to dihydroechinofuran.