Characterization of Disease-related 5β-Reductase (AKR1D1) Mutations Reveals Their Potential to Cause Bile Acid Deficiency
Characterization of Disease-related 5β-Reductase (AKR1D1) Mutations Reveals Their Potential to Cause Bile Acid Deficiency
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DOI：
10.1074/jbc.m110.127779
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发表时间：
2010-08-06
影响因子：
4.8
通讯作者：
Penning, Trevor M.
中科院分区：
文献类型：
--
作者：
Drury, Jason E.;Mindnich, Rebekka;Penning, Trevor M.
胆汁酸缺乏症是新生儿的一种严重综合症，如果不治疗可能导致死亡。5 β-还原酶缺乏症是胆汁酸缺乏症的一种形式，其特征在于具有生理活性的5 β-还原胆汁酸的水平显著降低。AKR 1D 1（醛酮还原酶1D 1）是唯一已知的立体特异性还原3-酮类固醇和甾醇中的Delta（4）双键以产生5 β-氢化产物的人类酶。对5名5 β-还原酶缺乏症患者的AKR 1D 1基因的分析显示，5种不同的突变导致蛋白质中的氨基酸取代。为了研究这些观察到的AKR 1D 1点突变在5 β-还原酶缺乏症中的因果作用，我们表征了它们对酶性质的影响。通过在大肠杆菌中过表达来纯化突变酶的尝试仅产生足够量的P133 R突变体用于进一步表征。该酶显示出高度降低的Km和V-max，这让人想起以4-异戊烯-7 α-醇-3-酮作为底物的非竞争性动力学。此外，该突变体显示辅因子亲和力没有变化，但在不存在NADPH的情况下更不耐热，如CD光谱所判断的。在HEK 293细胞中表达后比较所有突变体。尽管这些酶基于mRNA水平同等表达，但蛋白质表达和功能活性显著降低。环己酰亚胺处理还揭示了几种表达的突变体不太稳定。我们的研究结果表明，在5 β-还原酶患者中报告的AKR 1D 1突变导致活性酶水平显著降低，可能是胆汁酸缺乏综合征发生的原因。
Bile acid deficiency is a serious syndrome in newborns that can result in death if untreated. 5 beta-Reductase deficiency is one form of bile acid deficiency and is characterized by dramatically decreased levels of physiologically active 5 beta-reduced bile acids. AKR1D1 (aldo-keto reductase 1D1) is the only known human enzyme that stereo-specifically reduces the Delta(4) double bond in 3-keto steroids and sterols to yield the 5 beta-hydrogenated product. Analysis of the AKR1D1 gene in five patients with 5 beta-reductase deficiency revealed five different mutations resulting in an amino acid substitution in the protein. To investigate a causal role for these observed point mutations in AKR1D1 in 5 beta-reductase deficiency, we characterized their effect on enzymatic properties. Attempts to purify mutant enzymes by overexpression in Escherichia coli only yielded sufficient amounts of the P133R mutant for further characterization. This enzyme displayed a highly reduced K-m and V-max reminiscent of uncompetitive kinetics with 4-cholesten-7 alpha-ol-3-one as substrate. In addition, this mutant displayed no change in cofactor affinity but was more thermolabile in the absence of NADPH as judged by CD spectroscopy. All mutants were compared following expression in HEK 293 cells. Although these enzymes were equally expressed based on mRNA levels, protein expression and functional activity were dramatically reduced. Cycloheximide treatment also revealed that several of the expressed mutants were less stable. Our findings show that the reported mutations in AKR1D1 in patients with 5 beta-reductase lead to significantly decreased levels of active enzyme and could be causal in the development of bile acid deficiency syndrome.