Although flavin-dependent halogenases (FDHs) are attractive for C–H bond activation, their applications are limited due to low turnover and stability. We have previously shown that leakage of a halogenating intermediate, hypohalous acid (HOX), causes FDHs to be inefficient by lessening halogenation yield. Here we employed a mechanism-guided semi-rational approach to engineer the intermediate transfer tunnel connecting two active sites of tryptophan 6-halogenase (Thal). This Thal-V82I variant generates less HOX leakage and possesses multiple catalytic improvements such as faster halogenation, broader substrate utilization, and greater thermostability and pH tolerance compared with the wildtype Thal. Stopped-flow and rapid quench kinetics analyses indicated that rate constants of halogenation and flavin oxidation are faster for Thal-V82I. Molecular dynamics simulations revealed that the V82I substitution introduces hydrophobic interactions which regulate tunnel dynamics to accommodate HOX and cause rearrangement of water networks, allowing better use of various substrates than the wildtype. Our approach demonstrates that an in-depth understanding of reaction mechanisms is valuable for improving efficiency of FDHs.

尽管黄素依赖性卤化酶 (FDH) 对 C-H 键活化具有吸引力，但由于低周转率和稳定性，它们的应用受到限制。我们之前已经表明，卤化中间体次卤酸 (HOX) 的泄漏会通过降低卤化产率导致 FDH 效率低下。在这里，我们采用机制引导的半理性方法来设计连接色氨酸 6-卤素酶 (Thal) 的两个活性位点的中间传输通道。与野生型 Thal 相比，这种 Thal-V82I 变体产生更少的 HOX 泄漏并具有多项催化改进，例如更快的卤化、更广泛的底物利用以及更高的热稳定性和 pH 耐受性。停流和快速猝灭动力学分析表明，Thal-V82I 的卤化和黄素氧化的速率常数更快。分子动力学模拟表明，V82I 取代引入了疏水相互作用，调节隧道动力学以适应 HOX 并导致水网络重排，从而比野生型更好地利用各种底物。我们的方法表明，深入了解反应机制对于提高 FDH 的效率很有价值。