Steroid recognition and regulation of hormone action: crystal structure of testosterone and NADP+ bound to 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase.
BACKGROUND
Mammalian 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSDs) modulate the activities of steroid hormones by reversibly reducing their C3 ketone groups. In steroid target tissues, 3 alpha-HSDs act on 5 alpha-dihydrotestosterone, a potent male sex hormone (androgen) implicated in benign prostate hyperplasia and prostate cancer. Rat liver 3 alpha-HSD belongs to the aldo-keto reductase (AKR) superfamily and provides a model for mammalian 3 alpha-, 17 beta- and 20 alpha-HSDs, which share > 65% sequence identity. The determination of the structure of 3 alpha-HSD in complex with NADP+ and testosterone (a competitive inhibitor) will help to further our understanding of steroid recognition and hormone regulation by mammalian HSDs.


RESULTS
We have determined the 2.5 A resolution crystal structure of recombinant rat liver 3 alpha-HSD complexed with NADP+ and testosterone. The structure provides the first picture of an HSD ternary complex in the AKR superfamily, and is the only structure to date of testosterone bound to a protein. It reveals that the C3 ketone in testosterone, corresponding to the reactive group in a substrate, is poised above the nicotinamide ring which is involved in hydride transfer. In addition, the C3 ketone forms hydrogen bonds with two active-site residues implicated in catalysis (Tyr55 and His117).


CONCLUSIONS
The active-site arrangement observed in the 3 alpha-HSD ternary complex structure suggests that each positional-specific and stereospecific reaction catalyzed by an HSD requires a particular substrate orientation, the general features of which can be predicted. 3 alpha-HSDs are likely to bind substrates in a similar manner to the way in which testosterone is bound in the ternary complex, that is with the A ring of the steroid substrate in the active site and the beta face towards the nicotinamide ring to facilitate hydride transfer. In contrast, we predict that 17 beta-HSDs will bind substrates with the D ring of the steroid in the active site and with the alpha face towards the nicotinamide ring. The ability to bind substrates in only one or a few orientations could determine the positional-specificity and stereospecificity of each HSD. Residues lining the steroid-binding cavities are highly variable and may select these different orientations.
Bennett
M J
MJ
Department of Biochemistry and Biophysics, Johnson Research Foundation, Philadelphia, PA 19104-6059, USA.
Albert
R H
RH
Jez
J M
JM
Ma
H
H
Penning
T M
TM
Lewis
M
M
D000445:Aldehyde Oxidoreductases / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008322:Mammals; D008958:Models, Molecular; D009249:NADP / Q000737:chemistry* / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013256:Steroids / Q000378:metabolism; D013329:Structure-Activity Relationship; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism*
D013256:Steroids; D013739:Testosterone; D009249:NADP; D010088:Oxidoreductases; D000445:Aldehyde Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase