Journal of Lipid Research, Vol 35, 922-929, Copyright © 1994 by Lipid Research, Inc.
Purification and characterization of a novel 17 alpha-hydroxysteroid dehydrogenase from an intestinal Eubacterium sp. VPI 12708
P de Prada, KD Setchell and PB Hylemon
Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0678.
A novel steroid-inducible 17 alpha-hydroxysteroid dehydrogenase (17
alpha-HSDH) has been purified over 850-fold from an intestinal Eubacterium
sp. VPI 12708. The purified protein has a subunit molecular mass of 42,000
daltons and a native molecular weight (M(r)) of 160,000 as estimated by gel
filtration chromatography. Enzyme activity was induced by growth in the
presence of androstenedione or cholic acid, but not deoxycholic acid.
Enzymatic activity required anaerobic conditions and was highly specific
for NADP+ and the 17 alpha-hydroxy group of C-19 steroids. Estimated Km
values were 31 microM and 70 microM for androstenedione and
epitestosterone, respectively. Vmax values were estimated to be 3250
nmol/min per mg protein and 1800 nmol/min per mg protein for the reductive
and oxidative reactions, respectively. The pH optima for both the oxidative
and reductive reactions ranged between 5.5 and 7.0. Treatment with EDTA
completely inactivated 17 alpha-HSDH activity but activity was partially
restored by the addition of either Mg2+ (1 mM) or Zn2+ (10 mM). The
N-terminal amino acid sequence analysis of purified enzyme suggests that 17
alpha- HSDH may belong to a disulfide reductase gene family.
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