Unusual charge stabilization of NADP+ in 17β-hydroxysteroid dehydrogenase

Type 1 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1), a member of the short chain dehydrogenase reductase (SDR) family, is responsible for the synthesis of 17 beta-estradiol, the biologically active estrogen involved in the genesis and development of human breast cancers. Here, we report the crystal structures of the H221L 17 beta-HSD1 mutant complexed to NADP(+) and estradiol and the H221L mutant/NAD(+) and a H221Q mutant/estradiol complexes. These structures provide a complete picture of the NADP(+)-enzyme interactions involving the flexible 191-199 loop (well ordered in the H221L mutant) and suggest that the hydrophobic residues Phe(192)-Met(193) could facilitate hydride transfer. 17 beta-HSD1 appears to be unique among the members of the SDR protein family in that one of the two basic residues involved in the charge compensation of the 2'-phosphate does not belong to the Rossmann-fold motif. The remarkable stabilization of the NADP(+) 2'-phosphate by the enzyme also clearly establishes its preference for this cofactor relative to NAD(+). Analysis of the catalytic properties of, and estradiol binding to, the two mutants suggests that the His(221)-steroid O-3 hydrogen bond plays an important role in substrate specificity.