Crystal structures of mouse 17α-hydroxysteroid dehydrogenase (apoenzyme and enzyme-NADP(H) binary complex):: Identification of molecular determinants responsible for the unique 17α-reductive activity of this enzyme

Very recently, the mouse 17 alpha-hydroxysteroid dehydrogenase (m17 alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, has been characterized and identified as the unique enzyme able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta 4) into epitestosterone (epi-T), the 17 alpha-epimer of testosterone. Indeed, the other AKR enzymes that significantly reduce keto groups situated at position C17 of the steroid nucleus, the human type 3 3 alpha-HSD (h3 alpha-HSD3), the human and mouse type 5 17 beta-HSD, and the rabbit 20 alpha-HSD, produce only 17 beta-hydroxy derivatives, although they possess more than 70% amino acid identity with m17 alpha-HSD. Structural comparisons of these highly homologous enzymes thus offer an excellent opportunity of identifying the molecular determinants responsible for their 17 alpha/17 beta-stereospecificity. Here, we report the crystal structure of the m17 alpha-HSD enzyme in its apoform (1.9 angstrom resolution) as well as those of two different forms of this enzyme in binary complex with NADP(H) (2.9 angstrom and 1.35 angstrom resolution). Interestingly, one of these binary complex structures could represent a conformational intermediate between the apoenzyme and the active binary complex. These structures provide a complete picture of the NADP(H)enzyme interactions involving the flexible loop B, which can adopt two different conformations upon cofactor binding. Structural comparison with binary complexes of other AKR1C enzymes has also revealed particularities of the interaction between m17 alpha-HSD and NADP(H), which explain why it has been possible to crystallize this enzyme in its apo form. Close inspection of the m17 alpha-HSD steroid-binding cavity formed upon cofactor binding leads us to hypothesize that the residue at position 24 is of paramount importance for the stereospecificity of the reduction reaction. Mutagenic studies have showed that the m17 alpha-HSD(A24Y) mutant exhibited a completely reversed stereospecificity, producing testosterone only from Delta 4, whereas the h3 alpha-HSD3(Y24A) mutant acquires the capacity to metabolize Delta 4 into epi-T. (c) 2006 Elsevier Ltd. All rights reserved.