Purification and characterization of a novel dimeric 20 alpha-hydroxysteroid dehydrogenase from Tetrahymena pyriformis.
Tetrahymena pyriformis was found to exhibit high NADPH-dependent 20-oxosteroid reductase activity that converted 17 alpha-hydroxyprogesterone into 17 alpha,20 alpha-dihydroxypregn-4-en-3-one. The enzyme was purified 400-fold from the cytosolic fraction. The purified enzyme with a specific activity of 6.4 mumol/min per mg of protein had an isoelectric point of 4.9 and M(r) of 68,000, and was composed of two subunits of equal size. The N-terminal sequence was determined to be LAKTVPLNDGTNFPIFGG. The enzyme reduced pregnanes and pregnanes possessing a 17 alpha-hydroxy group to a greater extent than those without the hydroxy group, and oxidized 20 alpha-hydroxy groups of the steroids in the presence of NADP+. The Km values for 17 alpha-hydroxyprogesterone and 17 alpha-hydroxypregnenolone were 2.9 and 3.4 microM respectively. Although the enzyme was inactive towards androgens and oestrogens with 3- or 17-oxo groups, it reduced several nonsteroidal carbonyl compounds and oxidized trans-benzene dihydrodiol. The enzyme activity was inhibited by synthetic oestrogens, barbiturates, aldose reductase inhibitors and quercitrin. Thus, this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.149) which structurally and functionally differs from the mammalian and bacterial enzymes.
Inazu
A
A
Biochemistry Laboratory, Gifu Pharmaceutical University, Japan.
Sato
K
K
Nakayama
T
T
Deyashiki
Y
Y
Hara
A
A
Nozawa
Y
Y
D006907:17-alpha-Hydroxypregnenolone / Q000378:metabolism; D019326:17-alpha-Hydroxyprogesterone; D015089:20-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003600:Cytosol / Q000201:enzymology; D004795:Enzyme Stability; D006801:Humans; D006908:Hydroxyprogesterones / Q000378:metabolism; D007526:Isoelectric Point; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013769:Tetrahymena pyriformis / Q000201:enzymology*
D006908:Hydroxyprogesterones; D046911:Macromolecular Substances; D006907:17-alpha-Hydroxypregnenolone; D009249:NADP; D019326:17-alpha-Hydroxyprogesterone; D015089:20-Hydroxysteroid Dehydrogenases; D043607:20-alpha-Hydroxysteroid Dehydrogenase