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Structure/function aspects of human 3beta-hydroxysteroid dehydrogenase.

James L Thomas1, William L Duax, Anthony Addlagatta

  • 1Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, GA, USA.

Molecular and Cellular Endocrinology
|March 18, 2004
PubMed
Summary
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Separate genes encode human 3 beta-hydroxysteroid dehydrogenase/isomerase type 1 and type 2 (3 beta-HSD1, 3 beta-HSD2). Mutagenesis identified key catalytic residues and the structural basis (His156) for selective inhibition of 3 beta-HSD1.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Human 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) exists as two isoforms, 3 beta-HSD1 and 3 beta-HSD2, encoded by separate genes.
  • These isoforms play critical roles in steroidogenesis, with distinct tissue distributions (e.g., 3 beta-HSD1 in placenta and breast tumors, 3 beta-HSD2 in gonads and adrenal glands).
  • Understanding the catalytic mechanisms and structural differences between these isoforms is crucial for developing targeted therapeutic strategies.

Purpose of the Study:

  • To characterize the catalytic residues and functional differences between human 3 beta-HSD1 and 3 beta-HSD2.
  • To identify structural determinants responsible for substrate specificity and inhibitor affinity.
  • To explore the potential for selective inhibition of 3 beta-HSD1 for therapeutic applications.

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Main Methods:

  • Site-directed mutagenesis was employed to generate mutant enzymes of 3 beta-HSD1, targeting the putative catalytic motif and isomerase site.
  • Enzyme kinetics (Km values) were determined for wild-type and mutant enzymes using dehydroepiandrosterone (DHEA) as a substrate.
  • Inhibition kinetics were analyzed using Dixon plots to assess the affinity of epostane for different enzyme variants.
  • Homology modeling was used to predict the role of specific residues in cofactor binding and specificity.

Main Results:

  • Mutagenesis identified key catalytic residues, including Tyr154 and Lys158, essential for dehydrogenase activity, and Asp257/Asp258 for isomerase activity.
  • The H156Y mutation in 3 beta-HSD1 mimicked 3 beta-HSD2 kinetics, increasing substrate Km by 14-fold.
  • Epostane exhibited 17-fold greater inhibitory affinity for 3 beta-HSD1 compared to 3 beta-HSD2 and the H156Y mutant.
  • The D36A/K37R mutant shifted cofactor preference from NAD(H) to NADP(H).

Conclusions:

  • The study elucidated critical catalytic residues within 3 beta-HSD1, including His156, which dictates differences in substrate and inhibitor kinetics between isoforms.
  • The findings provide a structural basis for the differential inhibition of 3 beta-HSD1 and 3 beta-HSD2.
  • Selective inhibition of 3 beta-HSD1 presents a potential therapeutic strategy for hormone-sensitive breast tumors and managing placental steroidogenesis.