Flavin-Dependent Halogenases – From Cofactor Regeneration to Complex Substrates

黄素依赖性卤化酶（Flavin-dependent halogenases，FDH）仅需要黄素腺嘌呤二核苷酸（flavin adenine dinucleotide，FAD）、氧和卤化物盐来卤化它们的底物，并且因此提供了高度有吸引力的生物催化途径以高区域选择性地获得卤化化合物，这通常不能通过化学卤化来实现。该项目将联手剖析结构-功能关系，以工程师的活性，稳定性和底物范围的卤化酶。这将导致对选择性C-H激活的潜在机制和关键参数的更深入理解，如底物结合和识别以及辅因子再生。能够卤化肽内的芳香族氨基酸（Trp）的生物催化剂将被工程化以允许肽和-潜在地-甚至蛋白质卤化。为此，将合成色氨酸在不同位置的肽阵列，进行酶促卤化，并在进化活动中改进活性卤化酶。晶体结构将揭示活性位点中肽结合的结构基础。基于这些结构，我们将解决为什么只有某些卤化酶能有效地转化大底物的问题。辅因子再生将是这个项目的第二个重点。辅因子FAD需要以其完全还原状态（FADH 2）存在，以在第一催化步骤中与氧气反应。所有再生方案中的一个严峻挑战是游离FADH 2与氧的解偶联反应以产生过氧化氢，而不是结合到酶上进行催化。我们的目标是直接减少酶结合的FAD光化学，以避免解偶联，从而转化成一个有效的光驱动酶的卤化酶。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，我们将澄清的基本问题，是否FAD可以酶促再生，而它是绑定到卤化酶或是否部分或全部FAD需要从卤化酶解离转化为FADH 2的黄素reductase.In总结，我们计划揭示的要求卤化酶接受色氨酸含肽作为底物。我们还将区分赋予热稳定性的因素与影响活性的因素，并剖析辅助因子再生的机制，以直接提高卤化效率。此外，我们将澄清是否一个动态或共价结合的辅因子是有益的催化和研究蛋白质的灵活性在底物结合和营业额的作用。

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.