Cloning and expression of cDNA of human delta 4-3-oxosteroid 5 beta-reductase and substrate specificity of the expressed enzyme

Eur J Biochem. 1994 Jan 15;219(1-2):357-63. doi: 10.1111/j.1432-1033.1994.tb19947.x.

Abstract

The enzyme delta 4-3-oxosteroid 5 beta-reductase (3-oxo-5 beta-steroid: NADP+ oxidoreductase and 4,5 beta-dihydrocortisone: NADP+ delta 4-oxidoreductase) catalyzes the reduction of the delta 4 double bond of bile acid intermediates and steroid hormones carrying the delta 4-3-one structure in the A/B cis configuration. Human delta 4-3-oxosteroid 5 beta-reductase cDNA was isolated from a liver cDNA library by cross hybridization with a previously cloned rat cDNA, which was used as a probe [Onishi, Y. Noshiro, M., Shimosato, T. & Okuda, K.-I. (1991) FEBS Lett. 283, 215-218]. DNA sequence analysis of a hybridization-positive clone predicted the human delta 4-3-oxosteroid 5 beta-reductase to contain 326 amino acids. The amino acid sequence of the human delta 4-3-oxosteroid 5 beta-reductase had 79% overall identity to the rat enzyme sequence. It also showed 54% and 50% overall identity with rat 3 alpha-hydroxysteroid dehydrogenase and human aldose reductase, respectively. RNA blotting analysis demonstrated the existence of a single delta 4-3-oxosteroid 5 beta-reductase mRNA of approximately 2.7 kb in human liver. Transfection of the cDNA into COS cells resulted in the expression of an active enzyme with a high activity toward the bile acid intermediates 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one and 7 alpha-hydroxy-4-cholesten-3-one. In addition, the expressed enzyme showed a small but significant 5 beta-reduction activity toward 11 beta,17 alpha,21-trihydroxy-delta 4-pregnene-3,20-dione (cortisol) and 17 beta-hydroxy-delta 4-androsten-3-one (testosterone) whereas no activity was observed toward delta 4-pregnene-3,20-dione (progesterone) or delta 4-androstene-3-17-dione (androstenedione). The substrate specificity of the human enzyme is considerably narrower than that of the rat enzyme, and the enzyme seems to be more important for bile acid biosynthesis than for metabolism of steroid hormones.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Line
  • Cloning, Molecular
  • DNA, Complementary / biosynthesis
  • DNA, Complementary / isolation & purification
  • DNA, Complementary / metabolism
  • Escherichia coli
  • Gene Expression*
  • Humans
  • Immunoblotting
  • Liver / enzymology*
  • Molecular Sequence Data
  • Oxidoreductases / biosynthesis
  • Oxidoreductases / isolation & purification
  • Oxidoreductases / metabolism*
  • RNA / isolation & purification
  • RNA / metabolism
  • Rats
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Transfection

Substances

  • DNA, Complementary
  • Recombinant Proteins
  • RNA
  • Oxidoreductases
  • cholestenone 5 beta-reductase

Associated data

  • GENBANK/Z28339