A cinnamyl alcohol dehydrogenase (CAD) like enzyme leads to a branch in the shikonin biosynthetic pathway in Arnebia euchroma

Shikonin derivatives are natural naphthoquinone compounds and the main bioactive components produced by several boraginaceous plants, such as Lithospermum erythrorhizon and Arnebia euchroma. Phytochemical researches utilizing L. erythrorhizon and A. euchroma cultured cells both indicate the existence of a competing route branching out from the shikonin biosynthetic pathway toward benzo/hydroquinones. A previous study has shown that the branch point was a putative alcohol dehydrogenase converting (Z)-3''-hydroxygeranylhydroquinone ((Z)-3''-OH-GHQ) to an aldehyde intermediate ((Z)-3''-oxo-GHQ). However, the enzyme involved in the branch reaction was not characterized at the molecular level yet. In this study, we cloned a candidate gene belonging to the cinnamyl alcohol dehydrogenase (CAD) family, AeHGO， through coexpression analysis of transcriptome data sets of shikonin-proficient and shikonin-deficient cell lines of A. euchroma. In biochemical assays, purified AeHGO protein reversibly oxidizes (Z)-3''-OH-GHQ to produce (E)-3''-oxo-geranylhydroquinone ((E)-3''-oxo-GHQ) followed by reversibly reduce this product to (E)-3''-OH-GHQ, resulting in the coexistence of the three compounds. Time course analysis and kinetic parameters show that the reaction with (Z)-3''-OH-GHQ is about twice as efficient as with (E)-3''-OH-GHQ, which leads to the predominance of (E)-3''-OH-GHQ and (E)-3''-oxo-GHQ in the products. According to a previous report, (E)-3''-oxo-GHQ can be converted to deoxyshikonofuran, a hydroquinone metabolite produced by boraginaceous plants. Considering there is competition for the accumulation between shikonin derivatives and benzo/hydroquinones in both L. erythrorhizon and A. euchroma cultured cells, the reaction catalyzed by AeHGO could play an important role in the metabolic regulation of shikonin biosynthetic pathway. ### Competing Interest Statement The authors have declared no competing interest.