Isolation and characterization of S-adenosyl-L-methionine:Tetrahydroberberine-cis-N-methyltransferase from suspension cultures of Sanguinaria canadensis L

• Published in: Plant physiology (Bethesda) Vol. 105; no. 1; pp. 395 - 403
• Main Authors: O'Keefe, B.R, Beecher, C.W.W
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01-05-1994
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Agronomy. Soil science and plant productions; ALCALOIDE; ALCALOIDES; Alkaloids; Biological and medical sciences; BIOSINTESIS; BIOSYNTHESE; Biosynthesis; Chromatography; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; CULTIVO DE CELULAS; CULTURE DE CELLULE; Electrophoresis; Enzyme substrates; Enzymes; Fundamental and applied biological sciences. Psychology; Gels; Metabolism; Metabolism and Enzymology; METHIONINE; METIONINA; PAPAVERACEAE; Phosphates; Plant physiology and development; PROPIEDADES FISICO-QUIMICAS; PROPRIETE PHYSICOCHIMIQUE; PURIFICACION; PURIFICATION; REACCIONES QUIMICAS; REACTION CHIMIQUE; Sodium; Substrate specificity; TRANSFERASAS; TRANSFERASE; VIA BIOQUIMICA DEL METABOLISMO; VOIE BIOCHIMIQUE DU METABOLISME; Temperature; Purification; Enzyme; Transferases; Papaveraceae; Characterization; Alkaloid; Methyltransferases; Enzymatic activity; Isoelectric point; Dicotyledones; Substrate specificity; Angiospermae; Isolation; Spermatophyta
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.105.1.395

As part of a continuing study of the induction of alkaloid biosynthesis, we report the isolation to homogeneity and characterization of S-adenosyl-L-methionine: tetrahydroberberine-cis-N-methyl-transferase from suspension cultures of Sanguinaria canadensis that were induced to produce alkaloids by hormone depletion. This enzyme catalyzes the stereospecific transfer of a methyl group from S-adenosyl-L-methionine to the tertiary nitrogen of the protoberberine alkaloid tetrahydroberberine (canadine). The enzyme was purified 315-fold by ammonium sulfate precipitation, gel permeation chromatography, affinity dye chromatography, and both diethylaminoethyl and Mono-Q ion-exchange chromatography. The enzyme was further purified to an optimum specific activity of 225 nkat/mg of protein (3500-fold) and electrophoretic homogeneity by native polyacrylamide gel electrophoresis (PAGE). In contrast to previous reports with partially purified enzyme, the isolated protein was found to have a pH optimum of 7.0, a temperature optimum of 25 to 30 degrees C, and an isoelectric point of 5.1. Furthermore, the molecular weight of the homogeneous protein was found to be 39,000 by sodium dodecyl sulfate-PAGE. The homogeneous enzyme preferred tetrahydroberberine over all other substrates tested, showing an apparent Km of 2.1 micromolar, but also showed partial activity with tetrahydrojatrorrhizine and tetrahydropalmatrubine