SyrB2 in syringomycin E biosynthesis is a nonherne FeII α-ketoglutarate- and O2-dependent halogenase

The nine-residue lipodepsipeptide syringomycin E, elaborated as a phytotoxin by Pseudomonas syringae pv. syringae B301D contains a 4-Cl-L-Thr-9 moiety where failure to chlorinate results in a 3-fold drop in biological activity. The proteins SyrB1 and SyrB2 encoded by the biosynthetic cluster are shown to act as a substrate and enzyme pair for SyrB2-mediated chlorination of the aminoacyl-S-enzyme L-Thr-S-SyrB1. SyrB2 is a member of the nonheme Fe-II alpha-ketoglutarate-dependent enzyme superfamily, and requires 02 and a-ketoglutarate as well as chloride ion to carry out monochlorination of the -CH3 group of L-Thr-S-SyrB1. Chlorination of L-Thr-S-SyrB1 was validated by thioesterase-mediated release Of L-Thr and 4-Cl-L-Thr, N-derivatization as fluorescent isoindoles, and HPLC separation compared with authentic standards. Incubations with L-[C-14]Thr and [Cl-36(-)] as well as MS of the released products further validated identification. Enzymatic oxidative halogenation is a previously uncharacterized reaction type for nonheme Fell enzymes and may be the general mode for biosynthetic halogenation of aliphatic carbons of natural products.