Enabling regiospecific di-halogenation in one-pot reactions using an engineered single-component flavin-dependent tryptophan halogenase

Installation of halogen atoms to organic compounds through flavin-dependent halogenases (FDHs) catalyzed reactions has emerged as an attractive strategy in organic biochemistry and synthetic biology. A cyanobacterial FDH termed AetF that contains FDH and flavin reductase in the same polypeptide chain could become a useful tool enzyme as the need of including a reductase is omitted. Notably, AetF exploits a unique substrate-interaction network to consecutively brominate tryptophan (Trp) on C5 and then C7 to generate di-brominated Trp. In this study, structure-based engineering is used to eliminate the C7 halogenation capacity of AetF. The resulting variant AetF-AIF that contains three residue substitutions mainly catalyzes bromination or iodination on C5 of Trp. We also show that combining AetF-AIF in tandem with the wild type AetF enables the generation of heterogeneously halogenated Trp in a one-pot reaction. The final products, 5-Br-7-I-Trp or 5-I-7-Br-Trp depending on the addition order of halides, account for a high ratio in the reaction mixture without additional purification step. These results display the potentials of single-component Trp-FDHs in a wide application of the halogenation reactions. Table of Content Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/687318v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@101bd34org.highwire.dtl.DTLVardef@12ca797org.highwire.dtl.DTLVardef@aee280org.highwire.dtl.DTLVardef@1984907_HPS_FORMAT_FIGEXP M_FIG A single-component flavin-dependent halogenase that catalyzes di-bromination of tryptophan was engineered as a mono-halogenase and coupled in a one-pot reaction for the synthesis of heterogeneously halogenated tryptophan. C_FIG