To rationally engineer the substrate scope and selectivity of flavin-dependent halogenases (FDHs), it is essential to first understand the reaction mechanism and substrate interactions in the active site. FDHs have long been known to achieve regioselectivity through an electrophilic aromatic substitution at C7 of the natural substrate Trp, but the precise role of a key active-site Lys residue remains ambiguous. Formation of hypochlorous acid (HOCl) at the cofactor-binding site is achieved by the direct reaction of molecular oxygen and a single chloride ion with reduced FAD and flavin hydroxide, respectively. HOCl is then guided 10 Å into the halogenation active site. Lys79, located in this site, has been proposed to direct HOCl toward Trp C7 through hydrogen bonding or a direct reaction with HOCl to form an −NH₂Cl⁺ intermediate. Here, we present the most likely mechanism for halogenation based on molecular dynamics (MD) simulations and active-site density functional theory “cluster” models of FDH PrnA in complex with its native substrate l-tryptophan, hypochlorous acid, and the FAD cofactor. MD simulations with different protonation states for key active-site residues suggest that Lys79 directs HOCl through hydrogen bonding, which is confirmed by calculations of the reaction profiles for both proposed mechanisms.

中文翻译：

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黄素依赖性卤化酶的作用机制

为了合理设计黄素依赖性卤化酶 (FDH) 的底物范围和选择性，首先必须了解活性位点的反应机制和底物相互作用。人们早就知道 FDH 通过在天然底物 Trp 的 C7 处进行亲电芳族取代来实现区域选择性，但关键活性位点 Lys 残基的确切作用仍然不明确。在辅因子结合位点形成次氯酸 (HOCl) 是通过分子氧和单个氯离子分别与还原的 FAD 和氢氧化黄素直接反应实现的。然后将 HOCl 引导 10 Å 进入卤化活性位点。位于该位点的 Lys79 已被提议通过氢键或与 HOCl 直接反应形成-NH _{2将 HOCl 导向 Trp C7}Cl ⁺中间体。在这里，我们基于分子动力学 (MD) 模拟和 FDH PrnA 及其天然底物l-色氨酸、次氯酸和 FAD 辅因子复合物的活性位点密度泛函理论“簇”模型提出了最可能的卤化机制。关键活性位点残基具有不同质子化状态的 MD 模拟表明，Lys79 通过氢键引导 HOCl，这通过对两种提议机制的反应曲线的计算得到证实。