SyrB2 in syringomycin E biosynthesis is a nonheme FeII alpha-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 O2 and alpha-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-[14C]Thr and [36Cl-] 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.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Frédéric H
FH
Vaillancourt
Jun
J
Yin
Christopher T
CT
Walsh
D002712:Chlorides / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D005296:Ferrous Compounds / Q000378:metabolism; D005453:Fluorescence; D007656:Ketoglutaric Acids / Q000378:metabolism; D013058:Mass Spectrometry; D008956:Models, Chemical; D010100:Oxygen / Q000378:metabolism; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000096:biosynthesis*; D010957:Plasmids / Q000235:genetics; D044224:Pseudomonas syringae / Q000201:enzymology*
D002712:Chlorides; D017931:DNA Primers; D005296:Ferrous Compounds; D007656:Ketoglutaric Acids; D010456:Peptides, Cyclic; C105246:syringomycin E; D010453:Peptide Synthases; D010100:Oxygen