She Fukang, Anderson Brent W, Khana Daven B, Zhang Shenwei, Steinchen Wieland, Fung Danny K, Lesser Nathalie G, Lucas Lauren N, Stevenson David M, Astmann Theresa J, Bange Gert, van Pijkeren Jan-Peter, Amador-Noguez Daniel, Wang Jue D
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Department of Food Science, University of Wisconsin-Madison, Madison, Wisconsin, USA.
mSystems. 2025 Feb 18;10(2):e0113124. doi: 10.1128/msystems.01131-24. Epub 2025 Jan 28.
Gluconeogenesis, the reciprocal pathway of glycolysis, is an energy-consuming process that generates glycolytic intermediates from non-carbohydrate sources. In this study, we demonstrate that robust and efficient gluconeogenesis in bacteria relies on the allosteric inactivation of pyruvate kinase, the enzyme responsible for the irreversible final step of glycolysis. Using the model bacterium as an example, we discovered that pyruvate kinase activity is inhibited during gluconeogenesis via its extra C-terminal domain (ECTD), which is essential for autoinhibition and metabolic regulation. Physiologically, a mutant lacking the ECTD in pyruvate kinase displayed multiple defects under gluconeogenic conditions, including inefficient carbon utilization, slower growth, and decreased resistance to the herbicide glyphosate. These defects were not caused by the phosphoenolpyruvate-pyruvate-oxaloacetate futile cycle. Instead, we identified two major metabolic consequences of pyruvate kinase dysregulation during gluconeogenesis: failure to establish high phosphoenolpyruvate (PEP) concentrations necessary for robust gluconeogenesis and increased carbon overflow into the medium. analysis revealed that, in wild-type cells, an expanded PEP pool enabled by pyruvate kinase inactivation is critical for maintaining the thermodynamic feasibility of gluconeogenesis. Additionally, we discovered that exhibits glyphosate resistance specifically under gluconeogenic conditions, and this resistance depends on the PEP pool expansion resulting from pyruvate kinase inactivation. Our findings underscore the importance of allosteric regulation during gluconeogenesis in coordinating metabolic flux, efficient carbon utilization, and antimicrobial resistance.IMPORTANCEPyruvate kinase catalyzes the final irreversible step in glycolysis and is commonly thought to play a critical role in regulating this pathway. In this study, we identified a constitutively active variant of pyruvate kinase, which did not impact glycolysis but instead led to multiple metabolic defects during gluconeogenesis. Contrary to conventional understanding, these defects were not due to the phosphoenolpyruvate-pyruvate-oxaloacetate futile cycle. Our findings suggest that the defects arose from an insufficient buildup of the phosphoenolpyruvate pool and an increase in carbon overflow metabolism. Overall, this study demonstrates the essential role of pyruvate kinase allosteric regulation during gluconeogenesis in maintaining adequate phosphoenolpyruvate levels, which helps prevent overflow metabolism and enhances the thermodynamic favorability of the pathway. This study also provides a novel link between glyphosate resistance and gluconeogenesis.
糖异生是糖酵解的逆向途径，是一个耗能过程，可从非碳水化合物来源生成糖酵解中间产物。在本研究中，我们证明细菌中强大而高效的糖异生依赖于丙酮酸激酶的变构失活，丙酮酸激酶是负责糖酵解不可逆最后一步的酶。以模式细菌为例，我们发现丙酮酸激酶活性在糖异生过程中通过其额外的C末端结构域（ECTD）受到抑制，该结构域对于自身抑制和代谢调节至关重要。在生理上，丙酮酸激酶中缺乏ECTD的突变体在糖异生条件下表现出多种缺陷，包括碳利用效率低下、生长缓慢以及对除草剂草甘膦的抗性降低。这些缺陷不是由磷酸烯醇式丙酮酸 - 丙酮酸 - 草酰乙酸无效循环引起的。相反，我们确定了糖异生过程中丙酮酸激酶失调的两个主要代谢后果：未能建立强大糖异生所需的高磷酸烯醇式丙酮酸（PEP）浓度，以及碳向培养基中的溢流增加。分析表明，在野生型细胞中，丙酮酸激酶失活导致的PEP池扩大对于维持糖异生的热力学可行性至关重要。此外，我们发现该菌在糖异生条件下表现出对草甘膦的抗性，并且这种抗性取决于丙酮酸激酶失活导致的PEP池扩大。我们的研究结果强调了糖异生过程中变构调节在协调代谢通量、有效碳利用和抗微生物抗性方面的重要性。
重要性
丙酮酸激酶催化糖酵解的最后不可逆步骤，通常认为在调节该途径中起关键作用。在本研究中，我们鉴定了一种组成型活性丙酮酸激酶变体，它不影响糖酵解，但在糖异生过程中导致多种代谢缺陷。与传统认识相反，这些缺陷不是由于磷酸烯醇式丙酮酸 - 丙酮酸 - 草酰乙酸无效循环。我们的研究结果表明，这些缺陷源于磷酸烯醇式丙酮酸池积累不足和碳溢流代谢增加。总体而言，本研究证明了丙酮酸激酶变构调节在糖异生过程中维持足够的磷酸烯醇式丙酮酸水平的关键作用，这有助于防止溢流代谢并增强该途径的热力学有利性。本研究还提供了草甘膦抗性与糖异生之间的新联系。
