Flavin-Dependent Halogenases – From Cofactor Regeneration to Complex Substrates

黄素依赖性卤化酶（FDHs）只需要黄素腺嘌呤二核苷酸（FAD）、氧和卤化物盐来卤化它们的底物，因此提供了一种非常有吸引力的生物催化途径来获得具有高区域选择性的卤化化合物，这通常是化学卤化无法实现的。该项目将联手剖析结构-功能关系，以设计卤素酶的活性、稳定性和底物范围。这将使我们更深入地了解选择性C-H活化的潜在机制和关键参数，如底物结合和识别以及辅因子再生。能够在多肽内卤化芳香氨基酸（Trp）的生物催化剂将被设计成允许多肽甚至可能是蛋白质卤化。为此，将在不同位置合成具有色氨酸的肽阵列，进行酶卤化，并在进化运动中改进活性卤化酶。晶体结构将揭示肽在活性位点结合的结构基础。基于这些结构，我们将解决为什么只有某些卤化酶有效地转化大底物的问题。辅助因子再生将是该项目的第二个重点。辅助因子FAD需要以完全还原态（FADH2）存在，才能在催化的第一步与氧反应。在所有再生方案中，一个严峻的挑战是游离FADH2与氧解偶联反应生成过氧化氢，而不是与酶结合催化。我们的目标是直接光化学还原酶结合的FAD，以避免解偶联，从而将卤素酶转化为高效的光驱动酶。我们将通过向FAD引入共价键来解决FAD结合亲和力的问题。此外，我们将阐明FAD在与卤素酶结合时是否可以酶再生，或者部分或整个FAD是否需要与卤素酶分离才能通过黄素还原酶转化为FADH2的基本问题。总之，我们计划揭示卤化酶接受含色氨酸肽作为底物的要求。我们还将区分赋予热稳定性的因素和影响活性的因素，并剖析辅助因子再生直接提高卤化效率的机制。此外，我们将澄清辅助因子的动态或共价结合是否有利于催化，并研究蛋白质柔韧性在底物结合和周转中所起的作用。

Flavin-dependent halogenases (FDHs) require only flavin adenine dinucleotide (FAD), oxygen and halide salts for halogenating their substrates and thus provide a highly attractive biocatalytic access to halogenated compounds with high regioselectivity that often cannot be achieved by chemical halogenation. The project will join forces to dissect structure-function relationships in order to engineer halogenases for activity, stability, and substrate scope. This will lead to a deeper understanding of the underlying mechanism and the crucial parameters for selective C-H activation like substrate binding and recognition as well as cofactor regeneration. Biocatalysts capable of halogenating aromatic amino acids (Trp) within peptides will be engineered to allow for peptide and – potentially – even protein halogenation. For that purpose, arrays of peptides with tryptophan in different position will be synthesized, subjected to enzymatic halogenation, and active halogenases will be improved in evolution campaigns. Crystal structures will reveal the structural basis of peptide binding in the active site. Based on these structures we will address the question why only certain halogenases efficiently convert large substrates.Cofactor regeneration will be a second focus of this project. The cofactor FAD needs to be present in its fully reduced state (FADH2) to react in the first catalytic step with oxygen. A severe challenge in all regeneration schemes is the uncoupling reaction of free FADH2 with oxygen to give hydrogen peroxide instead of binding to the enzyme for catalysis. We aim at directly reducing the enzyme-bound FAD photochemically to avoid uncoupling and thereby converting the halogenase into an efficient light-driven enzyme. We will address the issue of FAD binding affinity by introducing a covalent bond to the FAD. Additionally, we will clarify the fundamental question of whether FAD can be enzymatically regenerated while it is bound to the halogenase or whether a part or the whole FAD needs to dissociate from the halogenase for conversion to FADH2 by a flavin reductase.In summary, we plan to reveal the requirements of halogenases for accepting Trp containing peptides as substrates. We will also distinguish factors conferring thermostability from factors influencing activity and dissect the mechanism of cofactor regeneration for direct improvements in halogenation efficiency. Moreover, we will clarify whether a dynamic or covalent binding of the cofactor is beneficial for catalysis and investigate the role protein flexibility plays in substrate binding and turnover.