Isolation and Characterization of S-Adenosyl-L-Methionine:Tetrahydroberberine-cis-N-Methyltransferase from Suspension Cultures of Sanguinaria canadensis L.
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-mehtyltransferase 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[deg]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 [mu]M, but also showed partial activity with tetrahydrojatrorrhizine and tetrahydropalmatrubine.
O'Keefe
B. R.
BR
Department of Medicinal Chemistry and Pharmacognosy, Program for Collaborative Research in the Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, Illinois 60612.
Beecher
CWW.
C