Purification and partial amino acid sequences of the enzyme vinorine synthase involved in a crucial step of ajmaline biosynthesis.
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 Mr for the enzyme of approximately 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.
Gerasimenko
Irina
I
Lehrstuhl für Pharmazeutische Biologie, Institut für Pharmazie, Johannes Gutenberg-Universität Mainz, Staudinger Weg 5, D-55099 Mainz, Germany.
Ma
Xueyan
X
Sheludko
Yuri
Y
Mentele
Reinhard
R
Lottspeich
Friedrich
F
Stöckigt
Joachim
J
D000101:Acetyl-CoA C-Acetyltransferase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D000404:Ajmaline / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D029062:Apocynaceae / Q000201:enzymology; D006822:Hybrid Cells / Q000201:enzymology; D026121:Indole Alkaloids; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011926:Rauwolfia / Q000201:enzymology; D020539:Sequence Analysis, Protein
D026121:Indole Alkaloids; C493776:vinorine; D000404:Ajmaline; D000101:Acetyl-CoA C-Acetyltransferase