HadA 单加氧酶中 Arg/Asn 对对 C4a-氢过氧-FAD 的形成和稳定,The FEBS Journal

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HadA 单加氧酶中 Arg/Asn 对对 C4a-氢过氧-FAD 的形成和稳定
The FEBS Journal ( IF 5.5 ) Pub Date : 2022-08-09 , DOI: 10.1111/febs.16591
Panu Pimviriyakul ₁ , Pimchai Chaiyen ₂

Affiliation

HadA 单加氧酶分别通过脱卤和脱硝催化卤代酚和硝基酚的解毒。C4a-氢过氧-FAD 是一种关键的反应性中间体，其形成、质子化和稳定性反映了酶的效率。在此，HadA 反应的瞬态动力学、定点诱变和 pH 依赖性行为被用于鉴定稳定 HadA 中 C4a 加合物的关键特征。C4a-氢过氧-FAD_的形成与 pH 无关，而其衰变和远端氧的质子化与 p Ka 有关值分别为 8.5 和 8.4。这些值与 7.6-9.1 的 pH 范围内的产物形成相关，表明加合物稳定对酶促效率的重要性。我们将 Arg101 鉴定为减少 FAD (FADH ^- ) 结合和 C4a-氢过氧-FAD 形成的关键残基，因为这些能力以及 HadA _R101A和 HadA _R101Q中酶活性的丧失。相邻 Asn447 的突变不影响 C4a-氢过氧-FAD 形成的速率；然而，它们会削弱 FADH ⁻结合。HadA N447A 中 Arg101/Asn447 氢键网络的破坏使 C4a-氢过氧-FAD衰变_的_p Ka值增加到 9.5；然而，这个 p K_a在 HadA _N447D中没有改变（p K _a为 8.5）。因此，Arg101/Asn447 对应该为 FADH ⁻结合提供重要的相互作用，并维持与从 HadA 中的 C4a-氢过氧-FAD 消除H ₂ O _{2相关的 p}K _{a 。}在底物存在的情况下，羟基化步骤中 C4a-羟基-FAD 的形成对 pH 不敏感，它脱水形成 p K _a为 7.9 的氧化 FAD。这种结构特征可能有助于阐明活性中间体在其他黄素依赖性单加氧酶中是如何稳定的。

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Formation and stabilization of C4a-hydroperoxy-FAD by the Arg/Asn pair in HadA monooxygenase

HadA monooxygenase catalyses the detoxification of halogenated phenols and nitrophenols via dehalogenation and denitration respectively. C4a-hydroperoxy-FAD is a key reactive intermediate wherein its formation, protonation and stabilization reflect enzyme efficiency. Herein, transient kinetics, site-directed mutagenesis and pH-dependent behaviours of HadA reaction were employed to identify key features stabilizing C4a-adducts in HadA. The formation of C4a-hydroperoxy-FAD is pH independent, whereas its decay and protonation of distal oxygen are associated with pK_a values of 8.5 and 8.4 respectively. These values are correlated with product formation within a pH range of 7.6–9.1, indicating the importance of adduct stabilization to enzymatic efficiency. We identified Arg101 as a key residue for reduced FAD (FADH⁻) binding and C4a-hydroperoxy-FAD formation due to the loss of these abilities as well as enzyme activity in HadA_R101A and HadA_R101Q. Mutations of the neighbouring Asn447 do not affect the rate of C4a-hydroperoxy-FAD formation; however, they impair FADH⁻ binding. The disruption of Arg101/Asn447 hydrogen bond networking in HadA_N447A increases the pK_a value of C4a-hydroperoxy-FAD decay to 9.5; however, this pK_a was not altered in HadA_N447D (pK_a of 8.5). Thus, Arg101/Asn447 pair should provide important interactions for FADH⁻ binding and maintain the pK_a associated with H₂O₂ elimination from C4a-hydroperoxy-FAD in HadA. In the presence of substrate, the formation of C4a-hydroxy-FAD at the hydroxylation step is pH insensitive, and it dehydrates to form the oxidized FAD with pK_a of 7.9. This structural feature might help elucidate how the reactive intermediate was stabilized in other flavin-dependent monooxygenases.

更新日期：2022-08-09

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