Biosystems and Integrative Sciences Institute, Faculdade de Ciências, Universidade de Lisboa, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.
Biosystems and Integrative Sciences Institute, Faculdade de Ciências, Universidade de Lisboa, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.
Biochim Biophys Acta Proteins Proteom. 2020 Jun;1868(6):140393. doi: 10.1016/j.bbapap.2020.140393. Epub 2020 Feb 19.
Multiple-CoA dehydrogenase deficiency (MADD) is an inborn disorder of fatty acid and amino acid metabolism caused by mutations in the genes encoding for human electron transfer flavoprotein (ETF) and its partner electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). Albeit a rare disease, extensive newborn screening programs contributed to a wider coverage of MADD genotypes. However, the impact of non-lethal mutations on ETF:QO function remains scarcely understood from a structural perspective. To this end, we here revisit the relatively common MADD mutation ETF:QO-p.Pro456Leu, in order to clarify how it affects enzyme structure and folding. Given the limitation in recombinant expression of human ETF:QO, we resort to its bacterial homologue from Rhodobacter sphaeroides (Rs), in which the corresponding mutation (p.Pro389Leu) was inserted. The in vitro biochemical and biophysical investigations of the Rs ETF:QO-p.Pro389Leu variant showed that, while the mutation does not significantly affect the protein α/β fold, it introduces some plasticity on the tertiary structure and within flavin interactions. Indeed, in the p.Pro389Leu variant, FAD exhibits a higher thermolability during thermal denaturation and a faster rate of release in temperature-induced dissociation experiments, in comparison to the wild type. Therefore, although this clinical mutation occurs in the ubiquinone domain, its effect likely propagates to the nearby FAD binding domain, probably influencing electron transfer and redox potentials. Overall, our results provide a molecular rational for the decreased enzyme activity observed in patients and suggest that compromised FAD interactions in ETF:QO might account for the known riboflavin responsiveness of this mutation.
多种酰基辅酶 A 脱氢酶缺乏症（MADD）是一种由编码人电子转移黄素蛋白（ETF）及其伴侣电子转移黄素蛋白：泛醌氧化还原酶（ETF：QO）的基因突变引起的脂肪酸和氨基酸代谢的先天性疾病。尽管这种疾病比较罕见，但广泛的新生儿筛查计划使得 MADD 基因型的覆盖面更广。然而，从结构角度来看，非致死性突变对 ETF：QO 功能的影响仍知之甚少。为此，我们重新研究了相对常见的 MADD 突变 ETF：QO-p.Pro456Leu，以阐明其如何影响酶的结构和折叠。鉴于重组表达人 ETF：QO 的局限性，我们求助于其来自球形红杆菌（Rs）的细菌同源物，其中插入了相应的突变（p.Pro389Leu）。对 Rs ETF：QO-p.Pro389Leu 变体的体外生化和生物物理研究表明，尽管该突变不会显著影响蛋白质的α/β折叠，但它会在三级结构和黄素相互作用中引入一些可塑性。实际上，在 p.Pro389Leu 变体中，与野生型相比，FAD 在热变性过程中表现出更高的热不稳定性，并且在温度诱导的解离实验中更快地释放。因此，尽管这种临床突变发生在泛醌结构域，但它的影响可能会传播到附近的 FAD 结合结构域，可能会影响电子转移和氧化还原电位。总的来说，我们的研究结果为患者中观察到的酶活性降低提供了分子依据，并表明 ETF：QO 中 FAD 相互作用的受损可能是该突变已知的核黄素反应性的原因。
