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

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• 作者：Fré ; dé ; rick Faucher ; Line Cantin ; Van Luu-The ; Fernand Labrie ; Rock Breton
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：August 12, 2008
• 年：2008
• 卷：47
• 期：32
• 页码：8261-8270
• 全文大小：425K
• 年卷期：v.47,no.32(August 12, 2008)
• ISSN：1520-4995

文摘

The 5β-reductases (AKR1D1−3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5β-reduction of the C4−C5 double bond found into Δ4-3-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, Glu₁₂₀, as the major molecular determinant for the unique activity displayed by 5β-reductases. To define the exact role played by this glutamate in the catalytic activity of these enzymes, biochemical and structural studies on human 5β-reductase (h5β-red) have been undertaken. The crystal structure of h5β-red in a ternary complex with NADP⁺ and 5β-dihydroprogesterone (5β-DHP), the product of the 5β-reduction of progesterone (Prog), revealed that Glu₁₂₀ 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 7α-hydroxy-4-cholesten-3-one and 7α,12α-dihydroxy-4-cholesten-3-one, when bound to the active site of h5β-red, can establish supplementary contacts with Tyr₂₆ and Tyr₁₃₂, two residues delineating the steroid-binding cavity. These additional contacts very likely account for the higher activity of h5β-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 h5β-red enzyme which lead to a reduction of its enzymatic activity and which can progress to severe liver function failure.