Increasing the Stability of Flavin-Dependent Halogenases by Disulfide Engineering

Flavin-dependent halogenases allow halogenation of electron-rich aromatic compounds under mild reaction conditions even at electronically unfavored positions with high regioselectivity. In order to expand the application of halogenases, the enzymes need to be improved in terms of stability and efficiency. A previous study with the tryptophan 6-halogenase Thal demonstrated that thermostable Thal variants tend to form dimers in solution while the wild type is present as a monomer. Based on this a dimeric Thal variant was generated that is covalently linked by disulfide bonds. Introducing two cysteine residues at the dimer interface resulted in the variant Thal CC with significantly increased thermostability (▵T₅₀ =15.7 K) and stability over time at elevated temperature compared to the wild type. By introducing the homologous mutations into the tryptophan 5-halogenase PyrH, we were able to show that the stabilization by covalent dimerization can also be transferred to other halogenases. Moreover, it was possible to further increase the thermostability of PyrH by inserting cysteine mutations at alternative sites of the dimer interface.