Department of Biochemistry, University of Missouri, Columbia, Missouri, USA.
Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA.
J Biol Chem. 2020 Jul 17;295(29):9901-9916. doi: 10.1074/jbc.RA120.012877. Epub 2020 May 27.
Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a "fly-casting mechanism." We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, mutant lines grown in a light-dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity.
乙酰辅酶 A 羧化酶（ACCase）催化脂肪酸合成的第一步。叶绿体中的多亚基 ACCase 在光适应时会因 pH 升高到 8 而被激活，而在暗适应时会因 pH 降低到 7 而被抑制。在此，通过对纯化的 ACCase 生物素连接结构域（BADC）和生物素羧基载体蛋白（BCCP）亚基进行滴定，表明它们可以独立地、高效地结合生物素羧化酶（BC），但对代表光或暗条件下质体基质中 pH 变化的响应不同。在磷酸盐缓冲液中 pH 为 7 时，BADC1 和 BADC2 比 BCCP1 和 BCCP2 对 BC 的亲和力更有优势。然而，在 KCl 溶液中 pH 为 8 时，BCCP1 和 BCCP2 对 BC 的亲和力比 BADC1 高 10 倍以上。BC 对 BCCP 和 BADC 伴侣的偏好的 pH 调节变化表明它们有助于异源 ACCase 的光依赖性调节。使用 NMR 光谱学，我们发现 BADC 和 BCCP 亚基在 pH 7 时的固有无序程度增加的证据。我们提出，这种固有无序可能通过“飞钓机制”促进与 BC 的快速结合。我们假设 pH 对 BADC 和 BCCP 亚基的影响会在夜间减弱 ACCase 的活性，而在白天增强其活性。与该假设一致，在光暗循环中生长的 突变株系在其种子中合成了更多的脂肪酸。总之，我们的研究结果提供了证据，证明 BADC 和 BCCP 亚基作为 pH 传感器发挥作用，是异源 ACCase 活性光依赖性切换所必需的。
