Department of Chemical and Environmental Engineering, University of California, Riverside, California, USA.
Department of Biochemistry, Molecular Biology & Biophysics, BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, USA.
Appl Environ Microbiol. 2024 Sep 18;90(9):e0060224. doi: 10.1128/aem.00602-24. Epub 2024 Aug 23.
The acetogen couples caffeate reduction with ferredoxin reduction and NADH oxidation via electron bifurcation, providing additional reduced ferredoxin for energy conservation and cell synthesis. Caffeate is first activated by an acyl-CoA synthetase (CarB), which ligates CoA to caffeate at the expense of ATP. After caffeoyl-CoA is reduced to hydrocaffeoyl-CoA, the CoA moiety in hydrocaffeoyl-CoA could be recycled for caffeoyl-CoA synthesis by an ATP-independent CoA transferase (CarA) to save energy. However, given that CarA and CarB are co-expressed, it was not well understood how ATP could be saved when both two competitive pathways of caffeate activation are present. Here, we reported a dual feedback inhibition of the CarB-mediated caffeate activation by the intermediate hydrocaffeoyl-CoA and the end-product hydrocaffeate. As the product of CarA, hydrocaffeate inhibited CarB-mediated caffeate activation by serving as another substrate of CarB with hydrocaffeoyl-CoA produced. It effectively competed with caffeate even at a concentration much lower than caffeate. Hydrocaffeoyl-CoA formed in this process can also inhibit CarB-mediated caffeate activation. Thus, the dual feedback inhibition of CarB, together with the faster kinetics of CarA, makes the ATP-independent CarA-mediated CoA loop the major route for caffeoyl-CoA synthesis, further saving ATP in the caffeate-dependent electron-bifurcating pathway. A genetic architecture similar to has been found in other anaerobic bacteria, suggesting that the feedback inhibition of acyl-CoA ligases could be a widely employed strategy for ATP conservation in those pathways requiring substrate activation by CoA.
This study reports a dual feedback inhibition of caffeoyl-CoA synthetase by two downstream products, hydrocaffeate and hydrocaffeoyl-CoA. It elucidates how such dual feedback inhibition suppresses ATP-dependent caffeoyl-CoA synthesis, hence making the ATP-independent route the main pathway of caffeate activation. This newly discovered mechanism contributes to our current understanding of ATP conservation during the caffeate-dependent electron-bifurcating pathway in the ecologically important acetogen . Bioinformatic mining of microbial genomes revealed contiguous genes homologous to within the genomes of other anaerobes from various environments, suggesting this mechanism may be widely used in other CoA-dependent electron-bifurcating pathways.
乙酰辅酶 A 合酶通过电子分叉将咖啡酸还原与铁氧还蛋白还原和 NADH 氧化偶联，为能量守恒和细胞合成提供额外的还原型铁氧还蛋白。咖啡酸首先被酰基辅酶 A 合成酶（CarB）激活，该酶在消耗 ATP 的情况下将 CoA 连接到咖啡酸上。咖啡酰辅酶 A 还原为羟基咖啡酰辅酶 A 后，羟基咖啡酰辅酶 A 中的 CoA 部分可通过非依赖 ATP 的辅酶 A 转移酶（CarA）循环用于咖啡酰辅酶 A 合成，以节省能量。然而，鉴于 CarA 和 CarB 是共表达的，当存在两种咖啡酸激活的竞争性途径时，如何节省 ATP 尚不清楚。在这里，我们报道了中间产物羟基咖啡酰辅酶 A 和终产物羟基咖啡酸对 CarB 介导的咖啡酸激活的双重反馈抑制。作为 CarA 的产物，羟基咖啡酸通过与产生的羟基咖啡酰辅酶 A 作为另一个 CarB 的底物来抑制 CarB 介导的咖啡酸激活。即使在远低于咖啡酸的浓度下，它也可以有效地与咖啡酸竞争。在此过程中形成的羟基咖啡酰辅酶 A 也可以抑制 CarB 介导的咖啡酸激活。因此，CarB 的双重反馈抑制与 CarA 更快的动力学一起，使非依赖 ATP 的 CarA 介导的辅酶 A 环成为咖啡酰辅酶 A 合成的主要途径，进一步节省了咖啡酸依赖性电子分叉途径中的 ATP。在其他厌氧菌中发现了类似于 的遗传结构，这表明酰基辅酶 A 连接酶的反馈抑制可能是那些需要 CoA 激活底物的途径中用于 ATP 保存的广泛采用的策略。
本研究报告了两种下游产物羟基咖啡酸和羟基咖啡酰辅酶 A 对咖啡酰辅酶 A 合成酶的双重反馈抑制。它阐明了这种双重反馈抑制如何抑制依赖 ATP 的咖啡酰辅酶 A 合成，从而使非依赖 ATP 的途径成为咖啡酸激活的主要途径。这种新发现的机制有助于我们了解在生态重要的乙酰辅酶 A 中，咖啡酸依赖性电子分叉途径中 ATP 的保存。微生物基因组的生物信息挖掘显示，来自各种环境的其他厌氧菌基因组中存在与 同源的连续基因，这表明该机制可能在其他依赖辅酶 A 的电子分叉途径中广泛使用。
