Unusual charge stabilization of NADP+ in 17beta-hydroxysteroid dehydrogenase.
Type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD1), a member of the short chain dehydrogenase reductase (SDR) family, is responsible for the synthesis of 17beta-estradiol, the biologically active estrogen involved in the genesis and development of human breast cancers. Here, we report the crystal structures of the H221L 17beta-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 Phe192-Met193 could facilitate hydride transfer. 17beta-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 His221-steroid O3 hydrogen bond plays an important role in substrate specificity.
Mazza
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C
Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de Biologie Structurale J.-P. Ebel, CEA-CNRS, 41, avenue des Martyrs, F-38027 Grenoble cedex, France.
Breton
R
R
Housset
D
D
Fontecilla-Camps
J C
JC
D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment
D009249:NADP; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase