Studies on steroid monooxygenase from Cylindrocarpon radicicola ATCC 11011. Purification and characterization.
A steroid monooxygenase from cells of a fungus, Cylindrocarpon radicicola ATCC 11011, grown in the presence of progesterone has been purified by affinity chromatography on a pregnenolone-Sepharose column. The obtained enzyme was gel electrophoretically homogeneous and exhibited a molecular weight of about 115,000. SDS-gel electrophoresis revealed that the enzyme consisted of two equal-sized subunits with a molecular weight of 56,000. Sedimentation equilibrium analysis at 20 degrees C indicated that the enzyme protein behaved as a mixture of monomeric and dimeric subunit species. The enzyme contained one molecule of FAD in each subunit and exhibited absorption maxima at 375 and 440 nm. The monooxygenase catalyzed a Baeyer-Villiger type oxidation, i.e., oxygenative esterification of C21-20-ketosteroid to form an acetate ester of C19-17 beta-hydroxysteroid with consumptions of NADPH and molecular oxygen. The enzyme displayed a wide substrate specificity toward C21-20-ketosteroids, while it strictly required NADPH as the external electron donor in a ratio of 1:1:1 for ketosteroid:NADPH:molecular oxygen. Kinetic study showed the enzyme to have very high affinity for progesterone.
D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology*; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D013053:Spectrophotometry; D013254:Steroid 17-alpha-Hydroxylase / Q000032:analysis / Q000302:isolation & purification*; D013250:Steroid Hydroxylases / Q000302:isolation & purification*; D013379:Substrate Specificity; D013696:Temperature
D009249:NADP; D013250:Steroid Hydroxylases; D013254:Steroid 17-alpha-Hydroxylase; C022445:progesterone 17-alpha-hydroxylase