The enzyme trans-4-Hydroxy-L-proline (Hyp) dehydratase (HypD) is among the most abundant glycyl radical enzymes (GREs) in the healthy human gut microbiome and is considered a promising antibiotic target for the prominent antibiotic-resistant pathogen Clostridium difficile. Although an enzymatic mechanism has been proposed, the role of the greater HypD protein environment in mediating radical reactivity is not well understood. To fill this gap in understanding, we investigate HypD across multiple time- and length- scales using electronic structure modeling and classical molecular dynamics. We observe that the Hyp substrate protonation state significantly alters both its enzyme-free reactivity and its dynamics within the enzyme active site. Accurate coupled cluster modeling suggests the deprotonated form of Hyp to be the most reactive protonation state for C5–Hpro-S activation. In the protein environment, hydrophobic interactions modulate the positioning of Cys434 radical to enhance the reactivity of C5–Hpro-S abstraction. Long-time dynamics reveal that changing Hyp protonation states triggers the switching of a Leu643-gated water tunnel, a functional feature that has not yet been observed for members of the GRE superfamily.

中文翻译：

---

蛋白质动力学和底物质子化状态介导反式-4-羟基-L-脯氨酸脱水酶的催化作用

反式4-羟基-L-脯氨酸（Hyp）脱水酶（HypD）是健康的人体肠道微生物组中最丰富的糖基自由基酶（GREs）之一，被认为是有前途的重要抗生素耐药性病原体梭状芽孢杆菌的抗生素靶标难。尽管已经提出了一种酶促机制，但是对于更大的HypD蛋白环境在介导自由基反应性中的作用还不甚了解。为了填补这一认识上的空白，我们使用电子结构模型和经典分子动力学研究了跨多个时间和长度尺度的HypD。我们观察到Hyp底物质子化状态显着改变其无酶反应性和酶活性位点内的动力学。准确的耦合簇模型表明，Hyp的去质子化形式是C5–Hpro-S活化最活跃的质子化状态。在蛋白质环境中，疏水相互作用调节Cys434自由基的位置，以增强C5-Hpro-S提取的反应性。长期的动力学表明，Hyp质子化状态的变化会触发Leu643门控水隧道的转换，这是GRE超家族成员尚未观察到的功能特征。