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Related Experiment Videos

Structure and function of 3 alpha-hydroxysteroid dehydrogenase

T M Penning1, M J Bennett, S Smith-Hoog

  • 1Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084, USA.

Steroids
|January 1, 1997
PubMed
Summary

Mammalian 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSDs) inactivate steroid hormones. Structural and mutagenesis studies reveal key catalytic residues and steroid-binding site determinants, aiding inhibitor design.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzymology

Background:

  • Mammalian 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSDs) are crucial for steroid hormone metabolism and receptor regulation.
  • 3 alpha-HSDs belong to the aldo-keto reductase (AKR) superfamily, sharing high sequence identity.
  • Rat liver 3 alpha-HSD is extensively characterized, with determined X-ray crystal structures providing structural templates.

Purpose of the Study:

  • To elucidate the structural and catalytic mechanisms of mammalian 3 alpha-HSDs.
  • To identify key amino acid residues involved in catalysis and steroid hormone binding.
  • To provide a basis for structure-based inhibitor design against 3 alpha-HSDs.

Main Methods:

  • X-ray crystallography to determine the apoenzyme and NADP+ complex structures of rat liver 3 alpha-HSD.

Related Experiment Videos

  • Site-directed mutagenesis of the catalytic tetrad (D50, Y55, K84, H117) and steroid-binding site residues (W86, W227).
  • Enzyme kinetics and binding assays (e.g., Kd measurements) to assess mutant enzyme activity and steroid binding affinity.

    • Main Results:

    • Rat liver 3 alpha-HSD adopts an (alpha/beta)8-barrel fold with NAD(P)(H) binding perpendicular to the axis.
    • A conserved catalytic tetrad (D50, Y55, K84, H117) and an apolar cleft form the active site for steroid binding.
    • Mutagenesis confirmed Y55 and K84 are essential for catalysis, while W86 and W227 are critical for steroid hormone recognition.
    • The Tyr/Lys catalytic dyad is conserved across AKR and short-chain dehydrogenase/reductase (SDR) families, suggesting convergent evolution.

    Conclusions:

    • The structural and mutagenesis data provide detailed insights into the catalytic mechanism and steroid-binding site of 3 alpha-HSDs.
    • Key residues like Y55, K84, W86, and W227 are critical for 3 alpha-HSD function.
    • Understanding these determinants enables rational design of inhibitors for therapeutic applications targeting steroid hormone pathways.