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 is a putative alcohol dehydrogenase converting (Z)-3-hydroxygeranylhydroquinone [(Z)-3-OH-GHQ] to an aldehyde intermediate (E)-3-oxo-GHQ. However, the enzyme involved in the branch reaction is not characterized at the molecular level yet. In this study, we clone 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-GHQ followed by reversibly reducing (E)-3-oxo-GHQ to (E)-3-OH-GHQ, resulting in an equilibrium mixture 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 equilibrium mixture. According to a previous report, (E)-3-oxo-GHQ can be converted to deoxyshikonofuran, a hydroquinone metabolite produced by boraginaceous plants. Considering there is a competition for accumulation between shikonin derivatives and benzo/hydroquinones in both L. erythrorhizon and A. euchroma cultured cells, AeHGO is supposed to play an important role in the metabolic regulation of shikonin biosynthetic pathway.