Application and Modification of Flavin-Dependent Halogenases

The application of flavin-dependent halogenases is hampered by their lack of stability under reaction conditions. However, first attempts to improve halogenase stability by error-prone PCR have resulted in mutants with higher temperature stability. To facilitate the screening for mutants with higher activity, a high-throughput assay was developed. Formation of cross-linked enzyme aggregates (CLEAs) of halogenases has increased halogenase lifetime by a factor of about 10, and CLEAs have been used to produce halogenated tryptophan in gram scale. Analyses of the substrate specificity of tryptophan halogenases have shown that they accept a much broader range of substrates than previously thought. The introduction of tryptophan halogenase genes into bacteria and plants led to the in vivo formation of peptides containing halogenated tryptophan or novel tryptophan-derived alkaloids, respectively. The halogen atoms in these compounds could be chemically exchanged against other substituents by cross-coupling reactions leading to novel compounds. Site-directed mutageneses have been used to modify the substrate specificity and the regioselectivity of flavin-dependent tryptophan halogenases. Since many flavin-dependent halogenases only accept protein-bound substrates, enzymatic and chemoenzymatic syntheses for protein-tethered substrates were developed, and the synthesized substrates were used in enzymatic halogenation reactions.