Phenylketonuria (PKU) is a genetic disorder that if left untreated can lead to behavioral problems, epilepsy, and even mental retardation. PKU results from mutations within the phenylalanine‐4‐hydroxylase (PAH) gene that encodes for the PAH protein. The study of all PAH causing mutations is improbable using experimental techniques. In this study, a collection of in silico resources, sorting intolerant from tolerant, Polyphen‐2, PhD‐SNP, and MutPred were used to identify possible pathogenetic and deleterious PAH non‐synonymous single nucleotide polymorphisms (nsSNPs). We identified two variants of PAH, I65N and L311P, to be the most deleterious and disease causing nsSNPs. Molecular dynamics (MD) simulations were carried out to characterize these point mutations on the atomic level. MD simulations revealed increased flexibility and a decrease in the hydrogen bond network for both mutants compared to the native protein. Free energy calculations using the MM/GBSA approach found that BH₄, a drug‐based therapy for PKU patients, had a higher binding affinity for I65N and L311P mutants compared to the wildtype protein. We also identify important residues in the BH₄ binding pocket that may be of interest for the rational drug design of other PAH drug‐based therapies. Lastly, free energy calculations also determined that the I65N mutation may impair the dimerization of the N‐terminal regulatory domain of PAH.

中文翻译：

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导致苯丙酮尿症遗传疾病的有害 PAH 突变的计算机筛选和分子动力学模拟

苯丙酮尿症 (PKU) 是一种遗传性疾病，如果不及时治疗会导致行为问题、癫痫甚至智力低下。PKU 由编码 PAH 蛋白的苯丙氨酸 4-羟化酶 (PAH) 基因内的突变引起。使用实验技术对所有引起 PAH 的突变进行研究是不可能的。在这项研究中，使用计算机资源的集合，将不耐受与耐受、Polyphen-2、PhD-SNP 和 MutPred 进行分类，以确定可能的致病性和有害的 PAH 非同义单核苷酸多态性 (nsSNP)。我们确定了 PAH 的两种变体，I65N 和 L311P，它们是最有害和引起疾病的 nsSNP。进行分子动力学 (MD) 模拟以在原子水平上表征这些点突变。MD 模拟显示，与天然蛋白质相比，两种突变体的灵活性增加，氢键网络减少。使用 MM/GBSA 方法的自由能计算发现 BH_{图 4}是 PKU 患者的一种基于药物的疗法，与野生型蛋白相比，它对 I65N 和 L311P 突变体具有更高的结合亲和力。我们还确定了 BH ₄结合口袋中的重要残基，这些残基可能对其他基于 PAH 药物的疗法的合理药物设计感兴趣。最后，自由能计算还确定 I65N 突变可能会损害 PAH N 端调节域的二聚化。