The crystal structure of progesterone 5β-reductase from Digitalis lanata defines a novel class of short chain dehydrogenases/reductases
The crystal structure of progesterone 5β-reductase from Digitalis lanata defines a novel class of short chain dehydrogenases/reductases
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DOI：
10.1074/jbc.m706185200
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发表时间：
2008-06-20
影响因子：
4.8
通讯作者：
Muller, Yves A.
中科院分区：
文献类型：
--
作者：
Thorn, Andrea;Egerer-Sieber, Claudia;Muller, Yves A.
孕酮 5β-还原酶 (5β-POR) 催化孕酮立体定向还原为 5β-孕烷-3,20-二酮，是洋地黄（毛地黄）植物中强心烯内酯生物合成途径的关键酶。序列考虑表明 5 beta-POR 是短链脱氢酶/还原酶 (SDR) 蛋白质家族的成员，但同时表明标准 SDR 中包含催化重要残基的序列基序缺失。在这里，我们展示了来自毛地黄的 5 β-POR 与 2.3 埃的 NADP(+) 复合物，且没有辅因子结合，分辨率为 2.4 埃，以及由 5 β-POR、NADP(+) 和黄体酮组成的三元复合物模型。事实上，5 beta-POR 显示了扩展 SDR 的折叠。然而，活性位点的结构是前所未有的，因为没有一个标准催化残基在结构上是保守的。活性位点中存在酪氨酸 (Tyr-179) 和赖氨酸残基 (Lys-147)，但它们从新位置展示并且是新序列基序的一部分。将 Tyr-179 突变为丙氨酸或苯丙氨酸会完全消除酶活性。我们认为，独特的拓扑结构反映了这样一个事实：5 beta-POR 通过 1-4 加成机制还原类固醇底物中的共轭双键，这需要重新定位催化重要残基。我们观察到，排列在活性位点上的序列基序在许多功能未知的细菌和植物酶中是保守的，这使我们得出这样的结论：5β-POR 定义了一类新的 SDR。
Progesterone 5 beta-reductase (5 beta-POR) catalyzes the stereospecific reduction of progesterone to 5 beta-pregnane-3,20-dione and is a key enzyme in the biosynthetic pathway of cardenolides in Digitalis (foxglove) plants. Sequence considerations suggested that 5 beta-POR is a member of the short chain dehydrogenase/reductase (SDR) family of proteins but at the same time revealed that the sequence motifs that in standard SDRs contain the catalytically important residues are missing. Here we present crystal structures of 5 beta-POR from Digitalis lanata in complex with NADP(+) at 2.3 angstrom and without cofactor bound at 2.4 angstrom resolution together with a model of a ternary complex consisting of 5 beta-POR, NADP(+), and progesterone. Indeed, 5 beta-POR displays the fold of an extended SDR. The architecture of the active site is, however, unprecedented because none of the standard catalytic residues are structurally conserved. A tyrosine (Tyr-179) and a lysine residue (Lys-147) are present in the active site, but they are displayed from novel positions and are part of novel sequence motifs. Mutating Tyr-179 to either alanine or phenylalanine completely abolishes the enzymatic activity. We propose that the distinct topology reflects the fact that 5 beta-POR reduces a conjugated double bond in a steroid substrate via a 1-4 addition mechanism and that this requires a repositioning of the catalytically important residues. Our observation that the sequence motifs that line the active site are conserved in a number of bacterial and plant enzymes of yet unknown function leads us to the proposition that 5 beta-POR defines a novel class of SDRs.