The purification and characterization of a unique cytochrome P-450 enzyme from Berberis stolonifera plant cell cultures.
A new cytochrome P-450 enzyme, isolated from Berberis stolonifera plant cell suspension cultures, has been purified to electrophoretic homogeneity. The purified hemoprotein migrated as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis with a minimal M(r) = 46,000. The enzyme could be purified to a high specific content of P-450 (18.2 nmol/mg protein) after fast protein liquid chromatofocusing, displaying an isoelectric point of 6.05. Spectral analysis of the homogeneous enzyme showed that it is predominantly low spin in the oxidized state, with a slight red-shifted ferrous carbonyl complex that exhibits a maximum at 452 nm. The purified cytochrome P-450, successfully reconstituted with NADPH-cytochrome P-450 reductase, displayed a maximal turnover rate of 50 nmol of substrate/nmol of P-450/min. In the purified and reconstituted form, the enzyme catalyzed the oxidation of three different chiral benzyltetrahydroisoquinoline substrates, namely (S)-coclaurine, (R)-N-methylcoclaurine, and (S)-N-methylcoclaurine, leading to the formation of three distinct dimeric products, (R,S)-berbamunine, (R,S)-2'-norberbamunine, and (R,R)-guattegaumerine, that are also present in the plant cell cultures in vivo. This is the first report of a P-450 enzyme that mediates regio- and stereoselective intermolecular oxidative phenol coupling to furnish natural dimeric compounds. In this catalytic cycle cytochrome P-450 functions as an oxidant in a bisubstrate reaction without transfer of the activated oxygen atom to either of the two chiral substrates.
Stadler
R
R
Lehrstuhl für Pharmazeutische Biologie, Universität München, Germany.
Zenk
M H
MH
D000470:Alkaloids / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism*; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D007546:Isoquinolines / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification* / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity; D014461:Ultracentrifugation
D000470:Alkaloids; D006882:Hydroxyapatites; D007546:Isoquinolines; D010636:Phenols; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D009251:NADPH-Ferrihemoprotein Reductase