Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) playing a pivotal role in the metabolism of strict and facultative anaerobes. Its activation is carried out by a PFL-activating enzyme, a member of the radical S-adenosylmethionine (rSAM) superfamily of metalloenzymes, which introduces a glycyl radical into the Gly radical domain of PFL. The activation mechanism is still not fully understood and is structurally based on a complex with a short model peptide of PFL. Here, we present extensive molecular dynamics simulations in combination with quantum mechanics/molecular mechanics (QM/MM)-based kinetic and thermodynamic reaction evaluations of a more complete activation model comprising the 49 amino acid long C-terminus region of PFL. We reveal the benefits and pitfalls of the current activation model, providing evidence that the bound peptide conformation does not resemble the bound protein–protein complex conformation with PFL, with implications for the activation process. Substitution of the central glycine with (S)- and (R)-alanine showed excellent binding of (R)-alanine over unstable binding of (S)-alanine. Radical stabilization calculations indicate that a higher radical stability of the glycyl radical might not be the sole origin of the evolutionary development of GREs. QM/MM-derived radical formation kinetics further demonstrate feasible activation barriers for both peptide and C-terminus activation, demonstrating why the crystalized model peptide system is an excellent inhibitory system for natural activation. This new evidence supports the theory that GREs converged on glycyl radical formation due to the better conformational accessibility of the glycine radical loop, rather than the highest radical stability of the formed peptide radicals.

中文翻译：

---

甘氨酰自由基酶的活化——多尺度建模洞察丙酮酸甲酸裂解酶活化酶的催化和自由基控制

丙酮酸甲酸裂解酶（PFL）是一种甘氨酰自由基酶（GRE），在严格和兼性厌氧菌的代谢中起关键作用。它的激活是由 PFL 激活酶进行的，该酶是金属酶自由基 S-腺苷甲硫氨酸 (rSAM) 超家族的成员，它将甘氨酰自由基引入 PFL 的 Gly 自由基结构域。激活机制仍未完全了解，并且在结构上基于具有 PFL 短模型肽的复合物。在这里，我们提出了广泛的分子动力学模拟，并结合基于量子力学/分子力学 (QM/MM) 的动力学和热力学反应评估，对包含 PFL 的 49 个氨基酸长 C 末端区域的更完整的激活模型进行了评估。我们揭示了当前激活模型的好处和缺陷，提供证据表明结合肽构象与 PFL 的结合蛋白质-蛋白质复合物构象不同，这对激活过程有影响。用(S)-和(R)-丙氨酸取代中心甘氨酸表明(R)-丙氨酸的结合优于(S)-丙氨酸的不稳定结合。自由基稳定性计算表明，甘氨酰自由基更高的自由基稳定性可能不是 GRE 进化发展的唯一来源。QM/MM 衍生的自由基形成动力学进一步证明了肽和 C 末端激活的可行激活障碍，说明了为什么结晶模型肽系统是一种极好的自然激活抑制系统。