Crystal Structures of Human Δ4-3-Ketosteroid 5β-Reductase (AKR1D1) Reveal the Presence of an Alternative Binding Site Responsible for Substrate Inhibition

The 5 beta-reductases (AKR1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 50-reduction of the C4-C5 double bond found in Delta 4-3-ketosteroids, including steroid hormones and bile acids precursors such as 7 alpha-hydroxy-4-cholesten-3-one and 7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one. In order to elucidate the binding mode and substrate specificity in detail, biochemical and structural studies on human 5 alpha-reductase (h5 beta-red; AKR1D1) have been recently undertaken. The crystal structure of a h5 beta-red binary complex provides a complete picture of the NADPH-enzyme interactions involving the flexible loop B, which contributes to the maintenance of the cofactor in its binding site by acting as a "safety belt". Structural comparison with binary complexes of AKR1C enzymes, specifically the human type 3 3 alpha-hydroxysteroid dehydrogenase (AKR1C2) and the mouse 17 alpha-hydroxysteroid dehydrogenase (AKR1C21), also revealed particularities in loop B positioning that make the steroid-binding cavity of h5 beta-red substantially larger than those of the two other enzymes. Kinetic characterization of the purified recombinant h5 beta-red has shown that this enzyme exerts a strong activity toward progesterone (Prog) and androstenedione (Delta 4) but is rapidly inhibited by these substrates once their concentrations reach 2-times their K-m value. A crystal structure of the h5 beta-red in ternary complex with NADPH and Delta 4 has revealed that the large steroid-binding site of this enzyme also contains a subsite in which the Delta 4 molecule is found. When bound in this subsite, Delta 4 completely impedes the passage of another substrate molecule toward the catalytic site. The importance of this alternative binding site for the inhibition of h5 beta-red was finally proven by site-directed mutagenesis, which demonstrated that the replacement of one of the residues delineating this site (Val(309)) by a phenylalanine completely abolishes the substrate inhibition. The results of this report provide structural insights into the substrate inhibition of h5 beta-red by C19- and C21-steroids.