Structure/function aspects of human 3β-hydroxysteroid dehydrogenase

Separate genes encode the human type 1 (placenta, breast tumors, other peripheral tissues) and type 2 (gonad, adrenal) isoforms of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD1, 3β-HSD2). Mutagenesis of 3β-HSD1 produced the Y154F, H156Y and K158Q mutant enzymes in the probable Y 154 -P-H 156 -S-K 158 catalytic motif. The H 156 Y mutant of the 3β-HSD1 created a chimera of the 3β-HSD2 motif (Y 154 -P-Y 156 -S-K 158 ) in 3β-HSD1. The D241N, D257L, D258L and D265N mutants are in the potential isomerase site of the 3β-HSD1 enzyme. Homology modeling with UDP-galactose-4-epimerase predicted that Asp 36 in the Rossmann-fold domain is responsible for the NAD(H) specificity of human 3β-HSD1, and our D36A/K37R mutant tested that assignment. The H 156 Y mutant of the 3β-HSD1 enzyme shifted the substrate (DHEA) kinetics to the 14-fold higher K m value measured for the 3β-HSD2 activity. From Dixon analysis, epostane inhibited the 3β-HSD1 activity with 17-fold greater affinity compared to 3β-HSD2 and H 156 Y. The mutants of Tyr 154 and Lys 158 exhibited no dehydrogenase activity and appear to be catalytic 3β-HSD residues. The D257L and D258L mutations eliminated isomerase activity, suggesting that Asp 257 or Asp 258 may be catalytic residues for isomerase activity. The D36A/K37R mutant shifted the cofactor preference of both 3β-HSD and isomerase from NAD(H) to NADP(H). In addition to characterizing catalytic residues, these studies have identified the structural basis (His 156 ) for an exploitable difference in the substrate and inhibition kinetics of 3β-HSD1 and 3β-HSD2. Hence, it may be possible to selectively inhibit human 3β-HSD1 to slow the growth of hormone-sensitive breast tumor cells and control placental steroidogenesis near term to prevent premature labor.