2019年,韩国高等科技学院的Ji-Joon Song团队在Nature communication上发表了关于双功能乙醛-乙醇脱氢酶(aldehyde-ethanol dehydrogenase, AdhE)形成的高阶螺旋体结构对其活性影响的文章。
AdhE是一种在细菌、真菌、藻类和原生动物中都高度保守的双功能醛醇脱氢酶,结构上由 N-末端乙醛脱氢酶(Aldehyde dehydrogenase,ALDH) 和 C-末端乙醇脱氢酶(Alcohol dehydrogenase, ADH) 2 个保守的结构域通过中间的Linker连接组成,分子量较大约为96kDa,且含有铁离子(如图1)。有研究发现,AdhE的寡聚体在电子显微镜下呈长丝状,也被称为螺旋体,其排列可能受到辅因子(NAD+和Fe2+)的影响。
图1 AdhE蛋白质结构
活性上具有两种脱氢酶活性:乙醛脱氢酶(ALDH)和乙醇脱氢酶(ADH),使得微生物在厌氧条件下可以将乙酰辅酶A通过两步反应转化为乙醇(如图2)。首先,N-端乙醛脱氢酶(ALDH)将乙酰辅酶A转化为乙醛,乙醛再由C-端乙醇脱氢酶(ADH)转化为乙醇。AdhE也是一种双向酶,因此也能以乙醇为底物来生成NADH和乙醛,再进一步通过ALDH催化生成乙酰辅酶A(如表1)。
图2 AdhE参与催化的反应
表1 AdhE的催化活性
虽然研究者们针对AdhE的研究日益深入,但对其高阶结构的分子细节以及这种结构对其酶活性的影响知之甚少。在该文章中,作者采用冷冻电镜,解析了AdhE的一个高分辨率(3.5埃)高阶螺旋体结构。在螺旋体结构中,ADH催化位点附近与另一个ADH分子之间产生螺旋间相互作用(图3b)。具体地说,是由来自两个ADH结构域的N492和R488残基之间产生的作用,且Q821能与来自另一个ADH结构域的残基816-821形成的loop产生相互作用。上述的相互作用空间附近存在NADH结合口袋。在螺旋体结构中,ALDH结构域以及相邻亚基的ADH结构域被聚集在一起,这可能使底物更容易到达两个酶的活性中心 (图3c)。为进一步研究AdhE的螺旋结构,在其结构上突出表示了ALDH和ADH可能的活性位点(图4d)。结果表明,ALDH活性中心位于螺旋结构的外表面,而ADH活性中心位于螺旋结构的内表面。由此证明,在AdhE螺旋体结构中,这两种酶的活性中心在拓扑上是分开的。
图3 AdhE螺旋体结构
b. 12个AdhE分子螺旋结构的冷冻电镜 (右)。原子模型显示了AdhE的两个半螺旋圈。红色方块表示ADH结构域之间螺旋间相互作用的区域。参与螺旋间相互作用的残基(R488、N492和Q821)如图所示。c. ALDH和ADH结构域分别为黄色和蓝色,表明结构域聚集(左侧视图和右上视图)。D模拟的NAD+辅因子的位置用空间填充表示, ALDH(红色)和ADH(蓝色)。e.12个(绿色)和24个(红色)分子在螺旋体形成的结构展示(右)
此外,作者发现ADH结构域中的F670残基插入到另一个ADH结构域中,并与其F462、I460和I712残基形成疏水口袋,从而将ADH-ADH结构域保持在一起(如图4)。将F670突变为Val(V)、Ala(A)和Glu(E),发现突变后的蛋白均未观察到螺旋结构(如图5),这表明F670介导的疏水相互作用对于螺旋体形成至关重要。
图4 ADH结构域之间的相互作用
图5 电镜下负染色后AdhE野生型及其突变体的蛋白结构
为探究螺旋体结构对AdhE活性的影响,作者对突变体的活性进行了检测。结果显示,突变体活性明显降低,说明螺旋体结构对维持活性至关重要(如图6)。
图6 AdhE野生型其突变体的酶活测定
该团队进一步探究了螺旋体在NADH存在下其螺旋结构的变化。在辅因子存在的情况下,作者给出了在辅因子存在的环境下,AdhE的高分辨率结构并计算其螺距及宽度的变化。结果显示在NAD+和FeSO4存在的情况下,AdhE的螺旋体结构似乎沿着长轴延伸,从而使ADHE的宽度变窄,螺距变长(如图7)。
图7 辅因子存在下AdhE的螺旋构象
综上所述,这项工作首次展示了AdhE的高阶螺旋体结构,并进一步证明了螺旋体结构对其维持其双功能活性的关键作用。
参考文献:
1.Aldehyde-alcohol dehydrogenase forms a high-order spirosome architecture critical for its activity. 2019, Nature Communication.
2.Pyruvate-formate-lyase-deactivase and acetyl-CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE.1991, FEBS Letters.
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