Faucher Frédérick, Cantin Line, Luu-The Van, Labrie Fernand, Breton Rock
Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL) and Laval University, Laval, Quebec (QC) G1V 4G2, Canada.
Biochemistry. 2008 Dec 23;47(51):13537-46. doi: 10.1021/bi801276h.
The 5beta-reductases (AKR1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5beta-reduction of the C4-C5 double bond found in Delta4-3-ketosteroids, including steroid hormones and bile acids precursors such as 7alpha-hydroxy-4-cholesten-3-one and 7alpha,12alpha-dihydroxy-4-cholesten-3-one. In order to elucidate the binding mode and substrate specificity in detail, biochemical and structural studies on human 5beta-reductase (h5beta-red; AKR1D1) have been recently undertaken. The crystal structure of a h5beta-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 3alpha-hydroxysteroid dehydrogenase (AKR1C2) and the mouse 17alpha-hydroxysteroid dehydrogenase (AKR1C21), also revealed particularities in loop B positioning that make the steroid-binding cavity of h5beta-red substantially larger than those of the two other enzymes. Kinetic characterization of the purified recombinant h5beta-red has shown that this enzyme exerts a strong activity toward progesterone (Prog) and androstenedione (Delta4) but is rapidly inhibited by these substrates once their concentrations reach 2-times their K(m) value. A crystal structure of the h5beta-red in ternary complex with NADPH and Delta4 has revealed that the large steroid-binding site of this enzyme also contains a subsite in which the Delta4 molecule is found. When bound in this subsite, Delta4 completely impedes the passage of another substrate molecule toward the catalytic site. The importance of this alternative binding site for the inhibition of h5beta-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 h5beta-red by C19- and C21-steroids.
5β-还原酶（AKR1D1 - 3）是一类独特的酶，能够高效且立体特异性地催化Δ4 - 3 - 酮类固醇（包括甾体激素和胆汁酸前体，如7α - 羟基 - 4 - 胆甾烯 - 3 - 酮和7α,12α - 二羟基 - 4 - 胆甾烯 - 3 - 酮）中C4 - C5双键的5β - 还原反应。为了详细阐明其结合模式和底物特异性，最近对人5β - 还原酶（h5β - red；AKR1D1）进行了生化和结构研究。h5β - 还原酶二元复合物的晶体结构完整呈现了NADPH与酶的相互作用，其中涉及柔性环B，该环通过充当“安全带”有助于将辅因子维持在其结合位点。与AKR1C酶的二元复合物（特别是人3型3α - 羟基类固醇脱氢酶（AKR1C2）和小鼠17α - 羟基类固醇脱氢酶（AKR1C21））的结构比较还揭示了环B定位的特殊性，这使得h5β - 还原酶的类固醇结合腔比另外两种酶的大得多。纯化的重组h5β - 还原酶的动力学特征表明，该酶对孕酮（Prog）和雄烯二酮（Δ4）具有很强的活性，但一旦这些底物的浓度达到其K(m)值的2倍，就会被迅速抑制。h5β - 还原酶与NADPH和Δ4的三元复合物的晶体结构表明，该酶的大类固醇结合位点还包含一个发现Δ4分子的亚位点。当Δ4结合在这个亚位点时，它会完全阻止另一个底物分子向催化位点的通过。最终通过定点诱变证明了这个替代结合位点对h5β - 还原酶抑制的重要性，该诱变表明用苯丙氨酸替换界定该位点的一个残基（Val(309)）会完全消除底物抑制。本报告的结果为C19和C21类固醇对h5β - 还原酶的底物抑制提供了结构上的见解。
