Flavin-Dependent Halogenases – From Cofactor Regeneration to Complex Substrates

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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.