The crystal structure of human Δ4-3-ketosteroid 5β-reductase defines the functional role of the residues of the catalytic tetrad in the steroid double bond reduction mechanism

The 5 beta-reductases (AKR1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5 beta-reduction of the C4-C5 double bond found into Delta 4-3-ketosteroids, including steroid hormones and bile acids. Multiple-sequence alignments and mutagenic studies have already identified one of the residues presumably located at their active site, Glu(120), as the major molecular determinant for the unique activity displayed by 5 beta-reductases. To define the exact role played by this glutamate in the catalytic activity of these enzymes, biochemical and structural studies on human 5 beta-reductase (h5 beta-red) have been undertaken. The crystal structure of h5 beta-red in a ternary complex with NADP(+) and 5 beta-dihydroprogesterone (5 beta-DHP), the product of the 5 beta-reduction of progesterone (Prog), revealed that Glu(120) does not interact directly with the other catalytic residues, as previously hypothesized, thus suggesting that this residue is not directly involved in catalysis but could instead be important for the proper positioning of the steroid substrate in the catalytic site. On the basis of our structural results, we thus propose a realistic scheme for the catalytic mechanism of the C4-C5 double bond reduction. We also propose that bile acid precursors such as 7 alpha-hydroxy-4-cholesten-3-one and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one, when bound to the active site of h5 beta-red, can establish supplementary contacts with Tyr(26) and Tyr(132), two residues delineating the steroid-binding cavity. These additional contacts very likely account for the higher activity of h5 beta-red toward the bile acid intermediates versus steroid hormones. Finally, in light of the structural data now available, we attempt to interpret the likely consequences of mutations already identified in the gene encoding the h5 beta-red enzyme which lead to a reduction of its enzymatic activity and which can progress to severe liver function failure.