Purification and characterization of a novel dimeric 20 alpha-hydroxysteroid dehydrogenase from Tetrahymena pyriformis

Biochem J. 1994 Jan 1;297 ( Pt 1)(Pt 1):195-200. doi: 10.1042/bj2970195.

Abstract

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.

Publication types

  • Comparative Study

MeSH terms

  • 17-alpha-Hydroxypregnenolone / metabolism
  • 17-alpha-Hydroxyprogesterone
  • 20-Hydroxysteroid Dehydrogenases / chemistry
  • 20-Hydroxysteroid Dehydrogenases / isolation & purification*
  • 20-Hydroxysteroid Dehydrogenases / metabolism
  • 20-alpha-Hydroxysteroid Dehydrogenase
  • Amino Acid Sequence
  • Animals
  • Catalysis
  • Cytosol / enzymology
  • Enzyme Stability
  • Humans
  • Hydroxyprogesterones / metabolism
  • Isoelectric Point
  • Macromolecular Substances
  • Molecular Sequence Data
  • Molecular Weight
  • NADP / metabolism
  • Oxidation-Reduction
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Tetrahymena pyriformis / enzymology*

Substances

  • Hydroxyprogesterones
  • Macromolecular Substances
  • 17-alpha-Hydroxypregnenolone
  • NADP
  • 17-alpha-Hydroxyprogesterone
  • 20-Hydroxysteroid Dehydrogenases
  • 20-alpha-Hydroxysteroid Dehydrogenase