Diversity Supplement R01GM139892 - Lorena Alamillo

项目总结 我们已经发现，单一的氢化物会导致内部结构和动力学的全局变化 一个酶家族的多个成员，在酶的结构、动力学和 对整个氧化还原酶超家族有影响的变构作用。具体来说，BLVRB家族是 NADPH依赖的还原酶存在于多种生物体中，它们通过 胆绿素还原为胆红素和广泛的黄素底物。虽然我们最近的出版物已经 揭示了辅酶结合与全球构象和动态变化的偶联，我们现在已经 发现有大范围的变化与辅酶的氧化状态相联系，直到23a. 离开。因此， 结构 催化作用 这一应用的中心前提是辅酶的氢化物全局偶联到这两个 和酶家族内的动态变化，并且这种全局耦合与 功能。 这里的新奇之处在于，我们将明确确定单一氢化物，即 NADPH/NADP+，是全球连接的(目标1)，以及这种全球耦合如何控制酶的功能(目标2)。进一步 创新包括以下几个方面。首先，我们发现氢化物偶联网络可以被调制 通过突变直接到酶/辅酶界面，也到远端偶联部位，这给了我们 确定这些网络在功能中的作用的独特机会。第二，我们发现， 进化变化的残基调制氢化物偶联网络和功能，提供了非凡的洞察力 氢化物介导的偶联和功能的进化作用。因此，进化上的差异将是 用于识别与辅酶的氧化状态偶联的变构网络，并同时揭示 它们在功能上的进化角色。基于我们的初步数据，包括核磁共振，X射线结晶学，和 生化研究，我们假设辅酶氧化会引起自身的构象变化，即 通过该酶在多个BLVRB家族成员中进一步全球传播(称为“InsideOut” 耦合)，并且耦合到这些变化的网络调制功能(被称为“in”耦合)。 我们将通过以下方式解决这一假设： 目标1)确定单一氢化物如何调节BLVRB内的全球动力学和结构 酶家族。将使用CSPs、松弛研究和系综方法进行核磁共振溶液研究 使用三种不同的方法来确定单一氢化物如何对动力学和结构进行全局调节 BLVRB家族中既有活性部位又有远端差异的成员(人、海兔和蚊子)。 目的2)确定与辅酶氧化状态偶联的网络的功能作用。 生物化学和生物物理方法将被用来确定氢化物介导的全球 调节，既包括与辅酶氢化物的直接相互作用的作用，也包括 连接到辅酶(变构)的通讯网络。

PROJECT SUMMARY We have discovered that a single hydride induces global changes to both structure and dynamics within multiple members of an enzyme family, providing a fundamental link between enzyme structure, dynamics, and allostery that has implications to the entire oxidoreductase superfamily. Specifically, the BLVRB family are NADPH-dependent reductases present in multiple organisms where they regulate cellular redox through the reduction of biliverdin-to-bilirubin and a wide array of flavin substrates. While our recent publications have revealed that coenzyme binding is coupled to global conformational and dynamic changes, we have now discovered that there are largescale changes coupled to the oxidation state of the coenzyme as far as 23 Å away. Thus, structural catalytic the central premise of this application is that a coenzyme's hydride is globally coupled to both and dynamic changes within an enzyme family and that such global coupling is integrally related to function. The novelty here is that we will explicitly determine how a single hydride, i.e., the difference between NADPH/NADP+, is globally linked (Aim 1) and how this global coupling controls enzyme function (Aim 2). Further innovation includes the following. First, we have discovered that hydride-coupled networks can be modulated by mutations directly to the enzyme/coenzyme interface but also to distally coupled sites, which gives us the unique opportunity to determine the role of these networks in function. Second, we have discovered that evolutionarily changing residues modulate hydride coupled networks and function, providing remarkable insight into the evolutionary role of hydride-mediated coupling and function. Evolutionary differences will therefore be exploited to identify allosteric networks coupled to the oxidative state of the coenzyme and simultaneously reveal their evolutionary roles in function. Based on our preliminary data that includes NMR, X-ray crystallographic, and biochemical studies, we hypothesize that the coenzyme oxidation induces its own conformational change that is further propagated globally through the enzyme in multiple BLVRB family members (referred to as “insideout” coupling) and that networks coupled to these changes modulate function (referred to as “outsidein” coupling). We will address this hypothesis through the following: Aim 1) Determine how a single hydride modulates the global dynamics and structure within the BLVRB family of enzymes. NMR solution studies using CSPs, relaxation studies, and ensembles methods will be used to determine how a single hydride imparts its global regulation to dynamics and structure using three distinct BLVRB family members with both active site and distal differences (human, hyrax, and mosquito). Aim 2) Determine the functional role of networks coupled to the oxidative state of the coenzyme. Biochemical and biophysical methods will be used to determine the functional role of hydride-mediated global regulation, which include both the role of direct interactions with the coenzyme's hydride as well as the role of networks of communication coupled to the coenzyme (allostery).