Departamento de Quimica Fisica, Facultad de Ciencias, Unidad de Excelencia de Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Universidad de Granada, Granada, Spain.
School of Biosciences, University of Kent, Canterbury, United Kingdom.
Mol Biol Evol. 2023 Mar 4;40(3). doi: 10.1093/molbev/msad032.
Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.
许多代谢物在一个生化途径的一步中产生，并在随后的步骤中消耗。这些代谢中间产物通常是反应性分子，如果允许它们在细胞内环境中自由扩散，可能会导致不必要的副反应，甚至对细胞产生毒性。因此，代谢中间产物通常被保护为与蛋白质结合的物质，并在多功能酶、多酶复合物和代谢物之间直接在酶活性位点之间转移。因此，将反应性代谢中间产物隔离有助于代谢效率。然而，目前尚不清楚这种进化适应是否可以在应对生物体生存挑战时放宽。在这里，我们报告了对大肠杆菌细胞的进化修复实验，其中一种对脯氨酸生物合成至关重要的酶已被删除。该删除使细胞无法在缺乏脯氨酸的培养基中生长。然而，令人惊讶的是，谷氨酰胺合成酶基因中的许多单一突变（至少 12 个）有效地恢复了细胞生长。这些突变导致一种高度反应性的磷酸化中间产物泄漏到细胞内环境中，这种中间产物是谷氨酰胺和脯氨酸生物合成途径共有的。通常认为这种中间产物仅以与蛋白质结合的形式存在。然而，突变诱导的泄漏使其扩散，从而为脯氨酸生物合成开辟了一条新途径。我们的结果支持这样一种观点，即隔离的代谢中间产物的泄漏很容易发生，并有助于某些情况下的生物体适应。反应性分子的可用性增加可能会为新的生化途径的产生提供条件，并注意到了代谢工程中突变诱导泄漏的潜力。
