Peptidic tryptophan halogenation by a promiscuous flavin-dependent enzyme

Amino acids undergo numerous enzymatic modifications, including free amino acids and those incorporated into polypeptides. The extensive structural modifications observed in ribosomally synthesized and post-translationally modified peptides (RiPPs) provide compelling examples of natural modifications to amino acids. However, the broad applicability of amino acid-modifying enzymes for synthetic purposes is limited by narrow substrate scope and often unknown regulatory or accessory factor requirements. Here, we characterize ChlH, a flavin-dependent halogenase (FDH) from the chlorolassin biosynthetic gene cluster. Reconstitution of ChlH and the associated flavin reductase ChlR in vitro unexpectedly showed that ChlH acts on the linear precursor peptide ChlA rather than the threaded lasso peptide product. Unlike characterized peptide-modifying FDHs, which are limited to either heavily altered peptidic scaffolds or the terminus of a linear peptide, ChlH halogenates internal Trp residues of linear peptides, as well as N- and C-terminal Trp. Ala and Trp scanning mutagenesis of ChlA revealed a broad substrate tolerance and the capacity to modify Trp at nearly every position of the core region. Halogenation of disparate RiPP precursor peptides, pharmacologically relevant peptides, and an internal Trp of a protein was achieved using wild-type ChlH. A rapid cell-free biosynthetic assay was also used to assess a mutagenic panel of ChlA, which provided insight into ChlH’s preferences. In contrast to characterized FDHs, ChlH halogenates diverse peptide sequences, and we predict this promiscuity may find utility in the modification of additional peptide and protein substrates of biotechnological value.