Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan.
Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan.
J Biol Chem. 2021 Sep;297(3):101027. doi: 10.1016/j.jbc.2021.101027. Epub 2021 Jul 31.
The FF synthase produces ATP from ADP and inorganic phosphate. The γ subunit of FF ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole FF complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire FF ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the β-hairpin structure of FF ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the β-hairpin structure, which provided novel insights into the regulatory mechanisms of FF ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.
FF 合酶可将 ADP 和无机磷酸转化为 ATP。光合生物中 FF ATP 合酶的γ亚基是该酶的转子亚基，其包含一个特征性的β发夹结构。该结构由插入序列形成，该序列仅在光养生物中保守。我们之前使用重组亚复合物证明，该区域在 ATP 水解活性的调节中发挥着重要作用，从而在响应光照环境变化时控制细胞内 ATP 水平。然而，该区域在 ATP 合成中的作用一直是一个悬而未决的问题，因为其分析需要制备整个 FF 复合物和跨膜质子动力。在这项研究中，我们成功地从蓝藻 Synechocystis sp. PCC 6803 制备了含有完整 FF ATP 合酶的蛋白脂质体，并测量了 ATP 合成/水解和质子转运活性。由于 Synechocystis 相对简单的遗传操作，使得对 FF ATP 合酶的β发夹结构及其活性的生化研究成为可能。我们进一步对缺失β发夹结构的 Synechocystis 突变株进行了生理分析，这为通过 γ 亚基中光养生物特有的区域提供了对蓝藻 FF ATP 合酶调节机制的新见解。我们的结果表明，该结构对 ATP 合成至关重要，并抑制 ATP 水解。
