Bacterial steroid monooxygenase catalyzing the Baeyer-Villiger oxidation of C21-ketosteroids from Rhodococcus rhodochrous: the isolation and characterization.
Steroid monooxygenase from Rhodococcus rhodochrous, isolated in homogeneity with a high yield, catalyzes Baeyer-Villiger oxidation of progesterone to produce testosterone acetate with the stoichiometric consumptions of NADPH and molecular oxygen. It is a flavoenzyme with the molecular size of 60 kDa in the monomeric form and the isoelectric point of 4.9. The absorption spectrum has the maxima at 278, 376, and 439 nm and the shoulders at 360 and 465 nm, indicating a strong hypsochromic shift (blue-shift) of the absorption peak in the visible wavelength region. The prosthetic group of the enzyme was identified to be FAD, and the Kd value was estimated to be 0.95 microM. The enzyme catalyzed only the oxidative esterification of progesterone, 11 alpha- and 11 beta-hydroxyprogesterone and not the oxidative lactonization of androstenedione. Km for progesterone was 100 microM, for NADPH was 3.3 microM, and the turnover number was 185 min-1. Kd values for progesterone, 11 alpha-hydroxyprogesterone, deoxycorticosterone, and androstenedione were 110, 130, 2000, and 450 microM, respectively. The optimum pH of the reaction was about 8.5. The reaction was inhibited competitively by 17 alpha-hydroxyprogesterone and androstenedione. Amino terminal sequences of the enzymes from the bacterium and also from fungus, Cylindrocarpon radiocicola were considerably different, and the potential flavin-binding site could be detected on the amino-terminal region of the fungus enzyme but not on that of the bacterial enzyme. Western blotting analyses of the two steroid monooxygenases resulted that mouse antiserum raised for each enzyme reacted only with the antigenic enzyme protein but did not show the cross-reactions. It is clarified that bacterial steroid monooxygenase is distinctly different from the fungal enzyme in the molecular and enzymic properties.
Miyamoto
M
M
Department of Chemistry, Faculty of Science, Kanazawa University, Japan.
Matsumoto
J
J
Iwaya
T
T
Itagaki
E
E
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D017666:Deuterium Oxide; D007664:Ketosteroids / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012240:Rhodococcus / Q000201:enzymology*; D013255:Steroid 21-Hydroxylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D013379:Substrate Specificity
D000596:Amino Acids; D007664:Ketosteroids; D013255:Steroid 21-Hydroxylase; D017666:Deuterium Oxide