Purification and properties of (S)-tetrahydroprotoberberine oxidase from suspension-cultured cells of Berberis wilsoniae.
A novel oxidase, catalyzing in the presence of oxygen the removal of four hydrogen atoms from a number of tetrahydroprotoberberines with simultaneous production of 1 mol H2O2 and H2O each, has been discovered and purified to homogeneity from Berberis wilsoniae cell cultures. This enzyme, (S)-tetrahydroprotoberberine oxidase, exhibited strict specificity for the (S)-enantiomer of tetrahydroprotoberberines and 1-benzylisoquinoline alkaloids, a pH optimum at 8.9, a molecular mass of 105 kDa and consisted of two subunits each of 53 kDa and covalently bound flavin. The Km values for (S)-scoulerine and (S)-norreticuline were 25 microM and 150 microM respectively. Concentration of the end-products, either protoberberines or H2O2, greater than 0.5 mM caused severe enzyme inhibition. This catalyst was responsible for the conversion of (S)-tetrahydrocolumbamine to the key intermediate, columbamine, in the metabolic pathway leading to berberine, jatrorrhizine and palmatine.
Amann
M
M
Lehrstuhl für Pharmazeutische Biologie, Universität München, Federal Republic of Germany.
Nagakura
N
N
Zenk
M H
MH
D001600:Berberine Alkaloids / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D010088:Oxidoreductases / Q000302:isolation & purification*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010946:Plants, Medicinal / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D001600:Berberine Alkaloids; C010941:discretamine; C055786:columbamine; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C056768:tetrahydroprotoberberine oxidase