Maurice Wilkins Centre, Ferrier Research Institute, Victoria University of Wellington, Wellington, New Zealand.
Maurice Wilkins Centre, Ferrier Research Institute, Victoria University of Wellington, Wellington, New Zealand.
J Biol Chem. 2021 Sep;297(3):101038. doi: 10.1016/j.jbc.2021.101038. Epub 2021 Jul 31.
Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bienzyme assembly comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactions of aromatic amino acid biosynthesis in the bacterium Prevotella nigrescens (PniDAH7PS), respectively. The interactions between these two distinct catalytic domains support functional interreliance within this bifunctional enzyme. The binding of prephenate, the product of CM-catalyzed reaction, to the CM domain is associated with a striking rearrangement of overall protein conformation that alters the interdomain interactions and allosterically inhibits the DAH7PS activity. Here, we have further investigated the complex allosteric communication demonstrated by this bifunctional enzyme. We observed allosteric activation of CM activity in the presence of all DAH7PS substrates. Using small-angle X-ray scattering (SAXS) experiments, we show that changes in overall protein conformations and dynamics are associated with the presence of different DAH7PS substrates and the allosteric inhibitor prephenate. Furthermore, we have identified an extended interhelix loop located in CM domain, loop, as a crucial segment for the interdomain structural and catalytic communications. Our results suggest that the dual-function enzyme PniDAH7PS contains a reciprocal allosteric system between the two enzymatic moieties as a result of this bidirectional interdomain communication. This arrangement allows for a complex feedback and feedforward system for control of pathway flux by connecting the initiation and branch point of aromatic amino acid biosynthesis.
模块化蛋白组装已被广泛报道为构建别构机制的一种机制。最近，在由 3-脱氧-D-阿拉伯庚酮糖-7-磷酸合酶（DAH7PS）和分支酸变位酶（CM）组成的双酶组装中，发现了一种独特的别构系统。这些酶分别催化细菌普雷沃特氏菌（PniDAH7PS）芳香族氨基酸生物合成的第一步和分支点反应。这两个不同催化结构域之间的相互作用支持了这种双功能酶内的功能相互依赖。预苯酸，即 CM 催化反应的产物，与 CM 结构域的结合与整体蛋白构象的惊人重排相关，该重排改变了结构域间相互作用，并别构抑制了 DAH7PS 的活性。在这里，我们进一步研究了这种双功能酶表现出的复杂别构通讯。我们观察到在存在所有 DAH7PS 底物的情况下，CM 活性的别构激活。通过小角 X 射线散射（SAXS）实验，我们表明整体蛋白构象和动力学的变化与不同的 DAH7PS 底物和别构抑制剂预苯酸的存在有关。此外，我们已经确定了位于 CM 结构域中的扩展的螺旋间环，loop，作为结构域间结构和催化通讯的关键片段。我们的结果表明，由于这种双向结构域间通讯，双功能酶 PniDAH7PS 在两个酶结构域之间包含一个相互的别构系统。这种排列允许通过连接芳香族氨基酸生物合成的起始和分支点，为途径通量的控制提供一个复杂的反馈和前馈系统。
