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

6533b833fe1ef96bd129b822

RESEARCH PRODUCT

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

Y. V. Sheludko Irina Gerasimenko Friedrich Lottspeich Reinhard Mentele Xueyan Ma Joachim Stöckigt

subject

Sequence analysis Stereochemistry Clinical Biochemistry Molecular Sequence Data Pharmaceutical Science Hybrid Cells Biochemistry Rauwolfia Indole Alkaloids chemistry.chemical_compound Vinorine synthase activity Biosynthesis Rauvolfia serpentina Sequence Analysis Protein Drug Discovery Amino Acid Sequence Acetyl-CoA C-Acetyltransferase Molecular Biology Peptide sequence chemistry.chemical_classification Ajmaline biology ATP synthase Molecular Structure Organic Chemistry Substrate (chemistry)biology.organism_classification Apocynaceae Enzyme Biochemistry chemistry biology.protein Molecular Medicine

description

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.

year	journal	country	edition	language
2004-01-13	Bioorganicmedicinal chemistry

10.1016/j.bmc.2004.02.028 https://pubmed.ncbi.nlm.nih.gov/15110859