Purification and characterization of dihydrobenzophenanthridine oxidase from elicited Sanguinaria canadensis cell cultures.
Upon treatment of Papaveracea cells with fungal elicitors, the biosynthesis of benzo[c]phenanthridine alkaloids is induced. Dihydrobenzophenanthridine oxidase, which catalyzes a later step in the biogenesis of these alkaloids, is one of the enzymes whose activity is elevated in the process. Here we report the 211-fold purification of the oxidase from elicited Sanguinaria canadensis by a combination of ammonium sulfate fractionation, DEAE-Sephadex, CM-Sephadex, Sephadex G-200, and either phenyl Superose or gel filtration chromatography. The purified enzyme utilized molecular oxygen to oxidize dihydrosanguinarine to sanguinarine with concomitant formation of hydrogen peroxide. A pH optimum of 7.0, Vmax of 27 nkat/mg protein, and apparent Km of 6.0 microM for dihydrosanguinarine were determined. Dihydrochelerythrine was also found to be a substrate for the purified enzyme, displaying an apparent Km of 10 microM. However, neither dihydronorsanguinarine nor the indole alkaloid ajmalicine was oxidized, indicating that the enzyme has some substrate specificity. Apparent molecular weight estimates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the most purified enzyme preparation obtained contained a major component of 77 kDa and two minor components between 59 and 67 kDa that can be associated with oxidase activity. Purified enzyme preparations possessed activity that was inhibited by sodium diethyldithiocarbamate, sodium azide, potassium cyanide, 1,4-DL-dithiothreitol, and mercaptoethanol.
Arakawa
H
H
E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104.
Clark
W G
WG
Psenak
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M
Coscia
C J
CJ
D002478:Cells, Cultured; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity
D010088:Oxidoreductases; C077698:dihydrobenzophenanthridine oxidase