Purification and partial amino acid sequences of the enzyme vinorine synthase involved in a crucial step of ajmaline biosynthesis

The isolation, enrichment, partial amino acid sequences and sequence comparison of vinorine synthase to other acetyltransferases from higher plants were reported. The acetyl-CoA-dependent enzyme vinorine synthase was isolated from hybrid cell suspension cultures of Rauvolfia serpentina and Rhazya stricta. The sarpagan-type alkaloid gardneral was used as a substrate of the enzyme leading to the ajmalan-type 10-methoxyvinorine. An HPLC-based assay was developed to monitor vinorine synthase activity, which allowed establishing a five step purification procedure combining anion exchange, hydrophobic interaction, hydroxyapatite and gel filtration. Purification resulted in a yield of 0.2% and an approximately 991-fold enrichment of the acetyltransfer activity. SDS-PAGE analysis showed a M r for the enzyme of ∼50 kDa. The four peptide fragments generated by proteolysis of the pure enzyme with endoproteinase LysC and the N-terminal part of the enzyme were sequenced. The enzyme preparation (>875-fold enrichment) delivering the N-terminal sequence was isolated from R. serpentina cell suspensions. Sequence alignment of the five peptides showed highest homologies in a range of 30–71% to acetyltransferases from other higher plants involved in natural plant product biosynthesis. Based on the partial sequences vinorine synthase is probably a novel member of the BAHD enzyme super family.