Peptidic tryptophan halogenation by a promiscuous flavin-dependent enzyme

Amino acids undergo numerous enzymatic modifications. 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. Unlike characterized peptide-modifying FDHs, which are limited to either specifically modified peptides or the termini of linear peptides, ChlH halogenates internal Trp residues of linear peptides, as well as N- and C-terminal Trp. Scanning mutagenesis of the substrate peptide ChlA revealed Trp was tolerated by ChlH at nearly every position. Molecular dynamics simulations corroborated the importance of a C-terminal motif in ChlA and provided insight into the lack of Trp14 chlorination in native chlorolassin. Furthermore, halogenation of disparate ribosomally synthesized and post-translationally modified peptide (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 provided insight into ChlHs 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. Entry for the Table of ContentsChlH, a flavin-dependent tryptophan halogenase, is reconstituted in vitro and found to be capable of modifying a wide array of diverse peptidic substrates despite showing selectivity on its native substrate peptide ChlA. This highlights its potential use in the biocatalytic production of chlorinated peptides. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/632611v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@897b21org.highwire.dtl.DTLVardef@1f2bafborg.highwire.dtl.DTLVardef@d1e800org.highwire.dtl.DTLVardef@d68381_HPS_FORMAT_FIGEXP M_FIG C_FIG