Stereospecific acetylation of shikonin and alkannin. (A) Phylogenetic analysis of LeSAT1 and LeAAT1 homologs of six Boraginaceae plants. Amino acid sequences were obtained from L. erythrorhizon (Le), E. plantagineum (Ep), A. euchroma (Ae), S. officinale (So), C. officinale (Co) and H. arborescens (Ha). Sequence alignment was performed using MAFFT, and a phylogenetic tree was generated using MEGA7 using the maximum-likelihood method. Bootstrap values based on 1,000 replicates are shown at the branching points. (B) Acetylation of shikonin and alkannin by SATs and AATs as analyzed by chiral HPLC. SAT1 and SAT2 mediated the acetylation of shikonin, whereas AAT1 and AAT2 mediated the acetylation of alkannin. Labeled compounds: 1, shikonin; 2, alkannin; 3, acetylalkannin; 4, acetylshikonin. (C) Reaction schemes of SATs and AATs.