Human steroid 5α-reductase 2 (SRD5A2) plays a determinative role in the masculinization of external genitalia. To date, approximately 114 different mutations of the SRD5A2 gene have been reported; however, little information is available about their impact on catalytic function or their three-dimensional (3D) structures. We determined the effect of point mutations on the testosterone-depend kinetic constants (Km,app and Vmax,app) and structural characteristics of SRD5A2 from Mexican patients with 46,XY-steroid 5α-reductase 2 deficiency. PCR-SSCP assays identified ten distinct gene variants and sequencing analysis identified missense mutations [p.V3I, p.S14R, p.A52T, p.F118L, p.R145W, p.R171S, p.L226P, p.F229S, p.S245Y, and p.A248V]. Mutations were re-created by site-directed mutagenesis and expressed in HEK293 cells. Functional studies demonstrated that 8 variants led to partial (Km,app = 0.16-2.6 μM; Vmax,app = 224-2640 pmol/mg P/min) or complete losses of activity compared to the wild-type enzyme (Km,app = 0.7 μM; Vmax,app = 4044 pmol/mg P/min). All the mutations were assessed using multiple software tools and the results predicted that all of the mutations were associated with disease or damage. Mapping mutations on the model of a 3D structure of SRD5A2 demonstrated alterations in contact sites with their proximal amino acids. Our data show that mutations affect the catalytic efficiency (Vmax/Km) or result in residual enzymatic activity, which could be due to erroneous interactions between amino acid residues, the substrate testosterone, or NADPH.

中文翻译：

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SRD5A2的突变分析：类固醇5α-还原酶2缺乏症的个体从基因到功能动力学。

人类固醇5α-还原酶2（SRD5A2）在外生殖器的男性化中起决定性作用。迄今为止，已经报道了SRD5A2基因的大约114种不同的突变；但是，有关它们对催化功能或它们的三维（3D）结构的影响的信息很少。我们确定了点突变对墨西哥患有46，XY-类固醇5α-还原酶2缺乏症患者的睾丸激素依赖性动力学常数（Km，app和Vmax，app）和SRD5A2结构特征的影响。PCR-SSCP分析鉴定出十种不同的基因变异，测序分析鉴定出错义突变[p.V3I，p.S14R，p.A52T，p.F118L，p.R145W，p.R171S，p.L226P，p.F229S，p。 S245Y和p.A248V]。通过定点诱变重新产生突变，并在HEK293细胞中表达。功能研究表明，与野生型酶相比，有8个变异导致部分（Km，app = 0.16-2.6μM; Vmax，app = 224-2640 pmol / mg P / min）或完全丧失活性（Km，app = 0.7μM； Vmax，app = 4044 pmol / mg P / min）。使用多种软件工具评估了所有突变，结果预测所有突变均与疾病或损害相关。SRD5A2的3D结构模型上的作图突变证明了其近端氨基酸接触位点的改变。我们的数据表明，突变会影响催化效率（Vmax / Km）或导致残留的酶活性，这可能是由于氨基酸残基，底物睾丸激素或NADPH之间的错误相互作用所致。app = 224-2640 pmol / mg P / min）或与野生型酶相比完全丧失活性（Km，app = 0.7μM; Vmax，app = 4044 pmol / mg P / min）。使用多种软件工具评估了所有突变，结果预测所有突变均与疾病或损害相关。SRD5A2的3D结构模型上的作图突变证明了其近端氨基酸接触位点的改变。我们的数据表明，突变会影响催化效率（Vmax / Km）或导致残留的酶活性，这可能是由于氨基酸残基，底物睾丸激素或NADPH之间的错误相互作用所致。app = 224-2640 pmol / mg P / min）或与野生型酶相比完全丧失活性（Km，app = 0.7μM; Vmax，app = 4044 pmol / mg P / min）。使用多种软件工具评估了所有突变，结果预测所有突变均与疾病或损害相关。SRD5A2的3D结构模型上的作图突变证明了其近端氨基酸接触位点的改变。我们的数据表明，突变会影响催化效率（Vmax / Km）或导致残留的酶活性，这可能是由于氨基酸残基，底物睾丸激素或NADPH之间的错误相互作用所致。使用多种软件工具评估了所有突变，结果预测所有突变均与疾病或损害相关。SRD5A2的3D结构模型上的作图突变证明了其近端氨基酸接触位点的改变。我们的数据表明，突变会影响催化效率（Vmax / Km）或导致残留的酶活性，这可能是由于氨基酸残基，底物睾丸激素或NADPH之间的错误相互作用所致。使用多种软件工具评估了所有突变，结果预测所有突变均与疾病或损害相关。SRD5A2的3D结构模型上的作图突变证明了其近端氨基酸接触位点的改变。我们的数据表明，突变会影响催化效率（Vmax / Km）或导致残留的酶活性，这可能是由于氨基酸残基，底物睾丸激素或NADPH之间的错误相互作用所致。