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 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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DOI：
10.1021/bi800572s
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发表时间：
2008-08-12
期刊：
影响因子：
2.9
通讯作者：
Breton, Rock
中科院分区：
文献类型：
--
作者：
Faucher, Frederick;Cantin, Line;Breton, Rock
5 β-还原酶（AKR 1D 1 -3）是能够以立体特异性方式有效催化C4-C5双键的5 β-还原成δ 4-3-酮类固醇（包括类固醇激素和胆汁酸）的独特酶。多序列比对和致突变研究已经确定了可能位于其活性位点的残基之一，Glu（120），作为5种β-还原酶显示的独特活性的主要分子决定因素。为了确定这种谷氨酸在这些酶的催化活性中所起的确切作用，对人类5 β-还原酶（h5 β-red）进行了生物化学和结构研究。5 β-红与NADP（+）和5 β-二氢孕酮三元配合物的晶体结构（5 β-DHP），孕酮（Prog）5 β-还原的产物，揭示了Glu（120）不直接与其他催化残基相互作用，如先前假设的那样，因此表明该残基不直接参与催化作用，而是对于甾族底物在催化位点的正确定位是重要的。在此基础上，我们提出了一个切实可行的C4-C5双键还原催化机理的方案。我们还提出，胆汁酸前体，如7 α-羟基-4-甾烯-3-酮和7 α，12 α-二羟基-4-甾烯-3-酮，当与h5 β-red的活性位点结合时，可以与Tyr（26）和Tyr（132）建立补充接触，这两个残基描绘了类固醇结合腔。这些额外的接触很可能是h5 β-red对胆汁酸中间体的活性高于类固醇激素的原因。最后，根据现有的结构数据，我们试图解释在编码h5 β-红酶的基因中已经确定的突变的可能后果，这些突变导致其酶活性降低，并可能进展为严重的肝功能衰竭。
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.