Purification and properties of codeinone reductase (NADPH) from Papaver somniferum cell cultures and differentiated plants.
Codeinone reductase (NADPH), which catalyzes the stereospecific reduction of (-)codeinone to (-)codeine, was detected and purified to electrophoretic homogeneity from a cytosolic fraction of Papaver somniferum L. cell cultures. The purification involved ammonium sulfate precipitation (40-80%), affinity chromatography (matrex red A), gel filtration (fractogel TSK HW 55S), affinity chromatography (fractogel TSK AF Blue), ion-exchange chromatography (DEAE-Sephacel) and native PAGE. The purified codeinone reductase was found to be a monomeric protein of 35 +/- 1 kDa that is highly substrate-specific, reducing only the C6 oxo group of codeinone and morphinone as well as a few analogues. The physiological forward reaction has a pH optimum at 7.0, the reverse reaction at 9.1. The temperature optimum is at 40 degrees C and the isoelectric point (p1) at 4.4. The apparent Km values (forward reaction) for codeinone and NADPH are 23 microM and 168 microM, respectively. Using capsule tissue of differentiated P. somniferum plants as an enzyme source, two codeinone reductase (NADPH) isoenzymes were detected and purified to homogeneity. These isoenzymes could not be separated for characterization and showed slightly different kinetic features (Km values: codeinone 9 microM; NADPH 81 microM) compared with the cell culture enzyme.
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Lehrstuhl für Pharmazeutische Biologie, Universität München, Germany.
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D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D003061:Codeine / Q000031:analogs & derivatives; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; D009249:NADP / Q000378:metabolism; D010207:Papaver / Q000201:enzymology*; D010946:Plants, Medicinal; D013045:Species Specificity; D013379:Substrate Specificity; D013696:Temperature
C046414:6-codeinone; D009249:NADP; D000429:Alcohol Oxidoreductases; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; C096678:codeinone reductase (NADPH); D003061:Codeine