PLANT PHYSIOLOGY , Vol 105, Issue 1 395-403, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Isolation and Characterization of S-Adenosyl-L-Methionine:Tetrahydroberberine-cis-N-Methyltransferase from Suspension Cultures of Sanguinaria canadensis L
B. R. O'Keefe and CWW. Beecher
Department of Medicinal Chemistry and Pharmacognosy, Program for Collaborative Research in the Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, Illinois 60612
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.
