Peptidic Tryptophan Halogenation by a Promiscuous Flavin‐Dependent Enzyme

作者：Andrew J. Rice, Mayuresh G. Gadgil, Paola Bisignano, Richard A. Stein, Hassane S. Mchaourab, Douglas A. Mitchell · 发表于：Angewandte Chemie International Edition · 年份：2025 · DOI：10.1002/anie.202509729 · 被引用次数：8 · 研究领域：Carbohydrate Chemistry and Synthesis、Fluorine in Organic Chemistry、Chemical Synthesis and Analysis

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 special examples of internal Trp within proteins was achieved using wild-type ChlH. A rapid cell-free biosynthetic assay 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.