Microbial degradation of the morphine alkaloids. Purification and characterization of morphine dehydrogenase from Pseudomonas putida M10.
The NADP(+)-dependent morphine dehydrogenase that catalyses the oxidation of morphine to morphinone was detected in glucose-grown cells of Pseudomonas putida M10. A rapid and reliable purification procedure involving two consecutive affinity chromatography steps on immobilized dyes was developed for purifying the enzyme 1216-fold to electrophoretic homogeneity from P. putida M10. Morphine dehydrogenase was found to be a monomer of Mr 32,000 and highly specific with regard to substrates, oxidizing only the C-6 hydroxy group of morphine and codeine. The pH optimum of morphine dehydrogenase was 9.5, and at pH 6.5 in the presence of NADPH the enzyme catalyses the reduction of codeinone to codeine. The Km values for morphine and codeine were 0.46 mM and 0.044 mM respectively. The enzyme was inhibited by thiol-blocking reagents and the metal-complexing reagents 1,10-phenanthroline and 2,2'-dipyridyl, suggesting that a metal centre may be necessary for activity of the enzyme.
Bruce
N C
NC
Institute of Biotechnology, University of Cambridge, U.K.
Wilmot
C J
CJ
Jordan
K N
KN
Stephens
L D
LD
Lowe
C R
CR
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000470:Alkaloids / Q000737:chemistry*; D002614:Chelating Agents / Q000737:chemistry; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D009022:Morphine Derivatives / Q000737:chemistry*; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000470:Alkaloids; D002614:Chelating Agents; D008670:Metals; D009022:Morphine Derivatives; D013438:Sulfhydryl Compounds; D000429:Alcohol Oxidoreductases; C045221:morphine 6-dehydrogenase