SyrB2 in syringomycin E biosynthesis is a nonheme Fe II α-ketoglutarate- and O 2 -dependent halogenase

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Abstract
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 α-ketoglutarate-dependent enzyme superfamily, and requires O 2 and α-ketoglutarate as well as chloride ion to carry out monochlorination of the -CH 3 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 -[ 14 C]Thr and [ 36 Cl - ] as well as MS of the released products further validated identification. Enzymatic oxidative halogenation is a previously uncharacterized reaction type for nonheme Fe II enzymes and may be the general mode for biosynthetic halogenation of aliphatic carbons of natural products.

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