Ciencia de los Alimentos, Centro de Investigación en Alimentación y Desarrollo A.C., Hermosillo, México.
Departamento de Ciencias Químico Biológicas y Agropecuarias, Universidad de Sonora Unidad Regional Sur, Navojoa, México.
J Mol Recognit. 2020 Oct;33(10):e2869. doi: 10.1002/jmr.2869. Epub 2020 Jun 22.
Betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine using NAD as a coenzyme. Porcine kidney BADH (pkBADH) follows a bi-bi ordered mechanism in which NAD binds to the enzyme before the aldehyde. Previous studies showed that NAD induces complex and unusual conformational changes on pkBADH and that potassium is required to maintain its quaternary structure. The aim of this work was to analyze the structural changes in pkBADH caused by NAD binding and the role played by potassium in those changes. The pkBADH cDNA was cloned and overexpressed in Escherichia coli, and the protein was purified by affinity chromatography using a chitin matrix. The pkBADH/NAD interaction was analyzed by circular dichroism (CD) and by isothermal titration calorimetry (ITC) by titrating the enzyme with NAD . The cDNA has an open reading frame of 1485 bp and encodes a protein of 494 amino acids, with a predicted molecular mass of 53.9 kDa. CD data showed that the binding of NAD to the enzyme caused changes in its secondary structure, whereas the presence of K helps maintain its α-helix content. K increased the thermal stability of the pkBADH-NAD complex by 5.3°C. ITC data showed that NAD binding occurs with different association constants for each active site between 37.5 and 8.6 μM. All the results support previous data in which the enzyme incubation with NAD provoked changes in reactivity, which is an indication of slow conformational rearrangements of the active site.
甜菜碱醛脱氢酶（BADH EC 1.2.1.8）以 NAD 为辅酶，催化甜菜碱醛不可逆氧化为甜菜碱。猪肾 BADH（pkBADH）遵循双顺序机制，其中 NAD 在醛之前结合到酶上。先前的研究表明，NAD 诱导 pkBADH 发生复杂而异常的构象变化，并且钾需要维持其四聚体结构。本工作旨在分析 NAD 结合引起的 pkBADH 结构变化以及钾在这些变化中所起的作用。克隆 pkBADH cDNA 并在大肠杆菌中过表达，然后使用 chitin 基质通过亲和层析纯化蛋白质。通过圆二色性（CD）和等温热滴定（ITC）分析 pkBADH/NAD 相互作用，通过用 NAD 滴定酶来滴定 NAD。cDNA 具有 1485bp 的开放阅读框，编码 494 个氨基酸的蛋白质，预测分子量为 53.9 kDa。CD 数据表明，NAD 与酶的结合导致其二级结构发生变化，而 K 的存在有助于维持其α-螺旋含量。K 使 pkBADH-NAD 复合物的热稳定性提高了 5.3°C。ITC 数据表明，NAD 结合在每个活性位点以不同的结合常数发生，范围在 37.5 和 8.6 μM 之间。所有结果都支持先前的数据，即酶与 NAD 孵育会引起反应性变化，这表明活性位点的缓慢构象重排。
