Doello Sofia, Shvarev Dmitry, Theune Marius, Sauerwein Jakob, Klon Alexander, Keskin Erva, Boehm Marko, Gutekunst Kirstin, Forchhammer Karl
Interfaculty Institute for Microbiology and Infection Medicine, Microbiology and Organismic Interactions, University of Tübingen, Tübingen 72076, Germany.
Department of Structural Biology, School of Biology/Chemistry, University of Osnabrück, Osnabrück 49076, Germany.
Proc Natl Acad Sci U S A. 2024 Dec 10;121(50):e2411604121. doi: 10.1073/pnas.2411604121. Epub 2024 Dec 6.
The oxidative pentose phosphate (OPP) pathway is a fundamental carbon catabolic route for generating reducing power and metabolic intermediates for biosynthetic processes. In addition, its first two reactions form the OPP shunt, which replenishes the Calvin-Benson cycle under certain conditions. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting reaction of this metabolic route. In photosynthetic organisms, G6PDH is redox-regulated to allow fine-tuning and to prevent futile cycles while carbon is being fixed. In cyanobacteria, regulation of G6PDH requires the redox protein OpcA, but the underlying molecular mechanisms behind this allosteric activation remain elusive. Here, we used enzymatic assays and in vivo interaction analyses to show that OpcA binds G6PDH under different environmental conditions. However, complex formation enhances G6PDH activity when OpcA is oxidized and inhibits it when OpcA is reduced. To understand the molecular basis of this regulation, we used cryogenic electron microscopy to determine the structure of G6PDH and the G6PDH-OpcA complex. OpcA binds the G6PDH tetramer and induces conformational changes in the active site of G6PDH. The redox sensitivity of OpcA is achieved by intramolecular disulfide bridge formation, which influences the allosteric regulation of G6PDH. In vitro assays reveal that the level of G6PDH activation depends on the number of bound OpcA molecules, which implies that this mechanism allows delicate fine-tuning. Our findings unveil a unique molecular mechanism governing the regulation of the OPP in .
氧化戊糖磷酸途径（OPP途径）是一条基本的碳分解代谢途径，用于产生生物合成过程所需的还原力和代谢中间体。此外，其前两个反应构成了OPP支路，在特定条件下可补充卡尔文-本森循环。葡萄糖-6-磷酸脱氢酶（G6PDH）催化该代谢途径的第一个反应且是限速反应。在光合生物中，G6PDH受到氧化还原调节，以便在碳固定过程中进行微调并防止无效循环。在蓝细菌中，G6PDH的调节需要氧化还原蛋白OpcA，但这种变构激活背后的潜在分子机制仍不清楚。在这里，我们通过酶活性测定和体内相互作用分析表明，OpcA在不同环境条件下与G6PDH结合。然而，当OpcA被氧化时，复合物的形成会增强G6PDH的活性，而当OpcA被还原时则会抑制其活性。为了理解这种调节的分子基础，我们使用低温电子显微镜来确定G6PDH以及G6PDH - OpcA复合物的结构。OpcA结合G6PDH四聚体并诱导G6PDH活性位点的构象变化。OpcA的氧化还原敏感性是通过分子内二硫键的形成实现的，这影响了G6PDH的变构调节。体外实验表明，G6PDH的激活水平取决于结合的OpcA分子数量，这意味着这种机制允许进行精细的微调。我们的研究结果揭示了一种独特的分子机制，该机制控制着蓝细菌中OPP途径的调节。
