Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
J Biol Chem. 2021 Dec;297(6):101423. doi: 10.1016/j.jbc.2021.101423. Epub 2021 Nov 18.
Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adenosyl-L-methionine (SAM) activase, is a simple and effective catalyst, but is also susceptible to oxidative damage in microaerobic environments. Such damage occurs at the glycyl radical cofactor, resulting in cleaved PFL (cPFL). Bacteria have evolved a spare part protein termed YfiD that can be used to repair cPFL. Previously, we obtained a structure of YfiD by NMR spectroscopy and found that the N-terminus of YfiD was disordered and that the C-terminus of YfiD duplicates the structure of the C-terminus of PFL, including a β-strand that is not removed by the oxygen-induced cleavage. We also showed that cPFL is highly susceptible to proteolysis, suggesting that YfiD rescue of cPFL competes with protein degradation. Here, we probe the mechanism by which YfiD can bind and restore activity to cPFL through enzymatic and spectroscopic studies. Our data show that the disordered N-terminal region of YfiD is important for YfiD glycyl radical installation but not for catalysis, and that the duplicate β-strand does not need to be cleaved from cPFL for YfiD to bind. In fact, truncation of this PFL region prevents YfiD rescue. Collectively our data suggest the molecular mechanisms by which YfiD activation is precluded both when PFL is not damaged and when it is highly damaged.
丙酮酸甲酸裂解酶（PFL）是一种甘氨酰基自由基酶（GRE），可将丙酮酸和辅酶 A 转化为乙酰辅酶 A 和甲酸盐，该反应对许多厌氧菌的初级代谢至关重要。甘氨酰基自由基辅因子是由自由基 S-腺苷甲硫氨酸（SAM）激活酶进行翻译后安装的，它是一种简单有效的催化剂，但在微氧环境中也容易受到氧化损伤。这种损伤发生在甘氨酰基自由基辅因子上，导致裂解的 PFL（cPFL）。细菌已经进化出一种备用蛋白 YfiD，可以用于修复 cPFL。以前，我们通过 NMR 光谱获得了 YfiD 的结构，并发现 YfiD 的 N 端无序，并且 YfiD 的 C 端重复了 PFL 的 C 端结构，包括氧诱导切割不除去的 β-链。我们还表明，cPFL 极易受到蛋白水解的影响，这表明 YfiD 对 cPFL 的拯救与蛋白降解竞争。在这里，我们通过酶学和光谱学研究来探究 YfiD 如何结合并恢复 cPFL 的活性的机制。我们的数据表明，YfiD 无规卷曲的 N 端区域对于 YfiD 甘氨酰基自由基的安装很重要，但对于催化作用不重要，并且对于 YfiD 结合来说，不需要从 cPFL 上切割重复的β-链。实际上，该 PFL 区域的截断阻止了 YfiD 的拯救。总的来说，我们的数据表明了 YfiD 激活被阻止的分子机制，既当 PFL 未受损时，也当 PFL 严重受损时。
