Faucher Frédérick, Pereira de Jésus-Tran Karine, Cantin Line, Luu-The Van, Labrie Fernand, Breton Rock
Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL) and Laval University, Québec (QC), Canada G1V 4G2.
J Mol Biol. 2006 Dec 8;364(4):747-63. doi: 10.1016/j.jmb.2006.09.030. Epub 2006 Sep 16.
Very recently, the mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-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 (Delta4) into epitestosterone (epi-T), the 17alpha-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 3alpha-HSD (h3alpha-HSD3), the human and mouse type 5 17beta-HSD, and the rabbit 20alpha-HSD, produce only 17beta-hydroxy derivatives, although they possess more than 70% amino acid identity with m17alpha-HSD. Structural comparisons of these highly homologous enzymes thus offer an excellent opportunity of identifying the molecular determinants responsible for their 17alpha/17beta-stereospecificity. Here, we report the crystal structure of the m17alpha-HSD enzyme in its apo-form (1.9 A resolution) as well as those of two different forms of this enzyme in binary complex with NADP(H) (2.9 A and 1.35 A 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 m17alpha-HSD and NADP(H), which explain why it has been possible to crystallize this enzyme in its apo form. Close inspection of the m17alpha-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 m17alpha-HSD(A24Y) mutant exhibited a completely reversed stereospecificity, producing testosterone only from Delta4, whereas the h3alpha-HSD3(Y24A) mutant acquires the capacity to metabolize Delta4 into epi-T.
最近，小鼠17α-羟基类固醇脱氢酶（m17α-HSD），醛酮还原酶（AKR）超家族的一员，已被表征并鉴定为能够高效且立体特异性地催化雄烯二酮（Δ4）转化为表睾酮（epi-T）的唯一酶，表睾酮是睾酮的17α-差向异构体。实际上，其他能显著还原类固醇核C17位酮基的AKR酶，人类3型3α-羟基类固醇脱氢酶（h3α-HSD3）、人类和小鼠5型17β-羟基类固醇脱氢酶以及兔20α-羟基类固醇脱氢酶，仅产生17β-羟基衍生物，尽管它们与m17α-HSD具有超过70%的氨基酸同一性。因此，对这些高度同源酶的结构比较为确定其17α/17β立体特异性的分子决定因素提供了绝佳机会。在此，我们报告了m17α-HSD酶无辅基形式的晶体结构（分辨率为1.9 Å）以及该酶与NADP(H)形成二元复合物的两种不同形式的晶体结构（分辨率分别为2.9 Å和1.35 Å）。有趣的是，这些二元复合物结构之一可能代表无辅基酶与活性二元复合物之间的构象中间体。这些结构提供了涉及柔性环B的NADP(H)-酶相互作用的完整图景，柔性环B在辅因子结合后可采取两种不同构象。与其他AKR1C酶二元复合物的结构比较还揭示了m17α-HSD与NADP(H)之间相互作用的特殊性，这解释了为何能够以无辅基形式结晶该酶。仔细观察辅因子结合后形成的m17α-HSD类固醇结合腔，使我们推测第24位残基对还原反应的立体特异性至关重要。诱变研究表明，m17α-HSD(A24Y)突变体表现出完全相反的立体特异性，仅从Δ4产生睾酮，而h3α-HSD3(Y24A)突变体获得了将Δ4代谢为epi-T的能力。
